Methods of treating lupus based on antibody affinity and screening methods and compositions for use thereof

ABSTRACT

The invention provides methods identifying individuals suitable for treatment for lupus and methods of monitoring treatment, based on measuring antibody affinities, as well as of treating lupus based on measuring antibody affinities. The treatment entails administration of a conjugate comprising a non-immunogenic valency platform molecule and at least two double stranded DNA epitopes, such as DNA molecules, which bind to anti-DNA antibodies from the patient.

RELATED APPLICATIONS

This application claims the priority benefit of U.S. provisionalapplication Ser. No. 60/167,716, filed Nov. 28, 1999, which isincorporated by reference in its entirety.

TECHNICAL FIELD

This invention relates to the field of antibody-mediated pathologiessuch as lupus. More particularly, the invention relates to methods oftreating individuals (and selecting individuals for treatment) for lupusbased on antibody affinity.

BACKGROUND ART

Systemic lupus erythematosus (SLE) is an autoimmune diseasecharacterized by the production of antibodies to a number of nuclearantigens, including double-stranded DNA (dsDNA). Autoantibodies thatreact with DNA are believed to play a role in the pathology of SLE andare closely associated with lupus nephritis. See, for example, Morimotoet al. (1982) J. Immunol. 139:1960-1965; Foster et al. (1993) Lab.Invest. 69:494-507; ter Borg et al. (1990) Arthritis Rheum. 33:634-643;Bootsma et al. (1995) Lancet 345:1595-1599.

Synthetic double-stranded oligonucleotides (dsON) have been shown tocross-react with anti-dsDNA antibodies (U.S. Pat. No. 5,276,013). Theuse of dsON conjugated with non-immunogenic carriers, also referred toas platforms, has been proposed for a therapeutic approach for thetreatment of SLE. For example, a tetrakis conjugate, LJP 249, composedof four dsON attached to a poly(ethylene glycol) valency platform wasused to demonstrate tolerance in an immunized mouse model system (Joneset al. (1994) Bioconjugate Chem. 5:390-399).

LJP 394, a tetravalent conjugate composed of four dsON attached to aplatform, was shown to delay progression of renal disease and extendsurvival in the BXSB experimental murine lupus nephritis model (Plunkettet al. (1995) Lupus 4:S99; Coutts et al. (1996) Lupus 5:158-159). LJP394 has also been shown to lower anti-dsDNA antibodies in human patientswith SLE (Weisman et al. (1997) J Rheumatol. 24:314-318).

Other literature describes methods which may be used in the treatment ofSLE, including methods of reducing levels of circulating antibodies byinducing B cell tolerance, including, but not limited to, U.S. Pat. Nos.5,276,013; 5,391,785; 5,786,512; 5,726,329; 5,552,391; 5,268,454;5,606,047; 5,633,395; 5,162,515; U.S. Ser. No. 08/118,055 (U.S. Pat. No.6,060,056); U.S. Ser. Nos. 60/088,656 and 60/103,088 (U.S. Ser. No.09/328,199 and PCT App. No. PCT/US99/13194).

Although overall patient prognosis in SLE has improved, treatmentregimens are not ideal and lupus nephritis continues to be associatedwith relatively poor overall survival (Seleznick et al. (1991) Semin.Arthritis Rheum. 21:73-80).

What is needed are improved methods of treatment of SLE and improvedmethods of identifying patients who may particularly respond to a giventreatment.

All references cited herein are hereby incorporated by reference intheir entirety.

DISCLOSURE OF THE INVENTION

The invention provides methods relating to immunotolerance treatment oflupus based on assessment of initial affinity of antibody from theindividual (i.e., antibody associated with lupus, namely, anti-ds DNAantibodies). The invention also provides methods of identifyingindividuals suitable (or unsuitable) for treatment based on assessingantibody affinity, as well as methods of treatment based on assessmentof change of affinity (if any) upon receiving treatment.

Accordingly, in one aspect, the invention provides methods of treatingSLE (including lupus nephritis) in an individual, comprisingadministering to the individual a conjugate comprising (a) anon-immunogenic valency platform molecule and (b) two or more doublestranded DNA epitopes, preferably polynucleotides, which specificallybind to an antibody from the individual which specifically binds todouble stranded DNA, wherein affinity of the epitope, preferablypolynucleotide, for the antibody from the individual is used as a basisfor selecting the individual to receive the treatment. In otherembodiments, the invention provides methods of treating SLE in anindividual, comprising administering to the individual a conjugatecomprising (a) a non-immunogenic valency platform molecule and (b) twoor more double stranded DNA epitopes, preferably polynucleotides whichspecifically bind to an antibody from the individual which specificallybinds to double stranded DNA, wherein affinity of the epitope,preferably polynucleotide, for the antibody from the individual is usedas a basis for selecting the individual to continue to receive thetreatment.

As using affinity as a basis for selecting an individual suitable fortreatment indicates, the treatment methods described herein generallyentail measuring antibody affinity (of an individual's anti-doublestranded DNA antibodies) for the dsDNA epitope.

In another aspect, the invention provides methods of treating systemiclupus erythematosus (SLE) in an individual, comprising administering tothe individual a conjugate comprising (a) a non-immunogenic valencyplatform molecule and (b) two or more polynucleotides which specificallybind to an antibody from the individual which specifically binds todouble stranded DNA, said polynucleotide preferably comprising,consisting essentially of or consisting of the double stranded DNAsequence 5′-GTGTGTGTGTGTGTGTGTGT-3′, wherein the apparent equilibriumdissociation constant (K_(D)′), or its functional equivalent, for thepolynucleotide with respect to the antibody from the individual beforeor upon initiation of treatment is less than about 1.0 mg IgG per ml,and wherein said K_(D)′ value (or its functional equivalent) is used asa basis for selecting the individual to receive the treatment.

In some embodiments, the treatment methods also include a selection stepcomprising assessing before initiation of treatment an apparentequilibrium dissociation constant (K_(D)′) (or its functionalequivalent) for the epitope, preferably a polynucleotide, containedwithin the conjugate with respect to antibodies from the individualwhich specifically bind to double stranded DNA, said conjugatecomprising (a) a non-immunogenic valency platform molecule and (b) twoor more dsDNA epitopes which specifically bind to an antibody from theindividual which specifically binds to double stranded DNA, wherein theindividual is selected to receive the treatment if the K_(D)′ (or itsfunctional equivalent) is less than about 1.0 mg IgG per ml. Other,lower K_(D)′ values are described herein which could apply to any of thedsDNA epitopes contemplated for use in treatment, as are percentileranking with respect to a given patient population as described herein.Preferably, the dsDNA epitopes are polynucleotides, said polynucleotidepreferably comprising, consisting essentially of or consisting of thedouble stranded DNA sequence 5′-GTGTGTGTGTGTGTGTGTGT-3′.

In another aspect, the invention provides methods of treating SLE in anindividual comprising: (a) assessing affinity of an anti-double strandedDNA antibody from the individual with respect to a dsDNA epitope whichis to be used in treatment, wherein the individual is selected fortreatment based on said antibody affinity; and (b) administering to saidselected individual a conjugate comprising (a) a non-immunogenic valencyplatform molecule and (b) two or more of the dsDNA epitopes.

In another aspect, the invention provides methods of treating lupusnephritis in an individual, comprising administering to the individual aconjugate comprising (a) a non-immunogenic valency platform molecule and(b) two or more polynucleotides which specifically bind to an antibodyfrom the individual which specifically binds to double stranded DNA,said polynucleotide comprising, consisting essentially of, or consistingof the double stranded DNA sequence 5′-GTGTGTGTGTGTGTGTGTGT-3′, whereinthe apparent equilibrium dissociation constant (K_(D)′) (or itsfunctional equivalent) for the polynucleotide in the conjugate withrespect to the antibody from the individual before or upon initiation oftreatment is less than about 1.0 mg IgG per ml, and wherein said K_(D)′value (or its functional equivalent) is used as a basis for selectingthe individual to receive the treatment.

In another aspect, the invention provides methods of treating SLE in anindividual, comprising: (a) assessing before or upon initiation oftreatment an apparent equilibrium dissociation constant (K_(D)′) for adsDNA epitope in or of a conjugate with respect to an antibody from theindividual which specifically binds to double stranded DNA, saidconjugate comprising (a) a non-immunogenic valency platform molecule and(b) two or more said epitopes which specifically bind to an antibodyfrom the individual which specifically binds to double stranded DNA and(b) administering to the individual the conjugate in an amountsufficient to increase the K_(D)′, wherein treatment is continued ifK_(D)′ is increased at least about 20% compared to K_(D)′ before or uponinitiation of treatment. In other embodiments, treatment methodscomprise administering any of the conjugate(s) described herein in anamount sufficient to reduce the affinity as reflected by an affinitymeasurement (as, for example, reflected by increased K_(D)′), preferablyby at least about 20%, although greater changes may be desirable.

In another aspect, the invention provides methods of treating lupusnephritis in an individual comprising administering to the individual aconjugate comprising (a) a non-immunogenic valency platform molecule and(b) two or more dsDNA epitopes which specifically bind to an antibodyfrom the individual which specifically binds to double stranded DNA. Insome embodiments, antibody affinity is assessed as described herein. Insome embodiments, conjugate is administered in an amount sufficient toreduce antibody affinity.

In another aspect, the invention provides methods of identifying anindividual who may be suitable for treatment for SLE, said treatmentcomprising administration of a conjugate comprising (a) anon-immunogenic valency platform molecule and (b) two or morepolynucleotides which specifically bind to an antibody from theindividual which specifically binds to double stranded DNA, saidpolynucleotide comprising, consisting essentially of, or consisting ofthe dsDNA sequence 5′-GTGTGTGTGTGTGTGTGTGT-3′, said method comprisingmeasuring the apparent equilibrium dissociation constant (K_(D)′) or itsfunctional equivalent for the polynucleotide used in the conjugate andanti-double stranded DNA antibodies from the individual before or uponinitiation of treatment, wherein an individual is identified by K_(D)′of less than about 1.0 mg IgG per ml or a functional equivalent thereof.

In another aspect, the invention provides methods of identifying anindividual who may be unsuitable for treatment for SLE, said treatmentcomprising administration of a conjugate comprising (a) anon-immunogenic valency platform molecule and (b) two or morepolynucleotides which specifically bind to an antibody from theindividual which specifically binds to double stranded DNA, saidpolynucleotide comprising, consisting essentially of, or consisting ofthe dsDNA sequence 5′-GTGTGTGTGTGTGTGTGTGT-3′, said method comprisingmeasuring the apparent equilibrium dissociation constant (K_(D)′) or itsfunctional equivalent for the polynucleotide of the conjugate andanti-double stranded DNA antibodies from the individual before or uponinitiation of treatment, wherein an individual is identified by K_(D)′of more than about 1.0 mg IgG per ml or a functional equivalent thereof.

In another aspect, the invention provides methods of monitoringtreatment for SLE in an individual, said treatment comprisingadministration of a conjugate comprising (a) a non-immunogenic valencyplatform molecule and (b) two or more dsDNA epitopes, preferablypolynucleotides, which specifically bind to an antibody from theindividual which specifically binds to double stranded DNA, said methodcomprising measuring the affinity for the dsDNA epitopes, preferablypolynucleotide(s) of the conjugate and anti-ds DNA antibodies from theindividual. In some aspects, the apparent equilibrium dissociationconstant (K_(D)′) is measured.

In another aspect, the invention provides kits comprising a molecule,such as a polynucleotide, comprising an epitope which binds to ananti-double stranded DNA antibody in suitable packaging, preferablyfurther comprising instructions as to measuring affinity of anti-ds DNAantibodies from an individual for the epitope.

In another aspect, the invention provides kits comprising (1) aconjugate comprising (a) a non-immunogenic valency platform molecule and(b) two or more polynucleotides which specifically bind to an antibodyfrom an individual which specifically binds to double stranded DNA; and(2) instructions for using the conjugate to detect affinity of theconjugate for anti-ds DNA antibodies from an individual.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C are graphs depicting competitive inhibition by LJP 394 ofantibodies from groups of SLE patients' sera binding to ¹²⁵I-labeleddsDNA.

FIG. 2 is a graph depicting the binding of sera from SLE patients andnormal patients by non-competitive affinity assay. IgG fraction from 10SLE serum samples and 10 normal serum samples was evaluated for bindingto the LJP 394 dsDNA epitope.

FIG. 3A is a graph depicting an apparent dose-dependent decrease inantibody affinity (as indicated by an increase in K_(D)′) in patientsreceiving 10 mg (solid circle) versus 50 mg (solid square) of conjugateLJP 394. Placebo is depicted as open circle. Visit 2 is pre-drugbaseline; visit 11 occurred after 4 months of weekly drugadministration. K_(D)′ values (Y-axis) are in mg/ml of IgG. FIG. 3B isthe same data depicted in the form of a bar graph.

FIGS. 4A and 4B are graphs depicting the K_(D)′ before LJP 394 treatmentor placebo (X-axis) versus the K_(D)′ after four months of LJP 394treatment or placebo (Y-axis). Graph A represents patients receivingtreatment with LJP 394 conjugate and graph B represents patientsreceiving placebo (i.e., not receiving treatment). FIGS. 4C and 4D aregraphs depicting the same data presented in FIGS. 4A and 4B with theaddition of data from additional patients.

FIGS. 5A and 5B are graphs depicting change in K_(D)′ in patientsreceiving conjugate LJP 394 (A, open squares) and in patients receivingplacebo (B, solid squares). Each square represents a patient. X-axis isthe K_(D)′ before receiving treatment (or placebo); Y-axis is thepercentage change in K_(D)′ after 4 months of treatment (or placebo).

FIGS. 6A and 6B are graphs which depict data the same data as that shownin FIGS. 5A and 5B with the addition of data from additional patients.The two panels depict the change in Kd′ in patients receiving conjugatesLJP 394 (left panel) or placebo (right panel). Each square represents anindividual patient. X-axis is the Kd′ before receiving treatment (orplacebo); Y-axis is the percentage change in K_(D)′ after 4 months oftreatment with 100 mg LJP 394 (or placebo) i.v. weekly.

FIG. 7 is a graph depicting a summary of the effects of drug treatmenton the affinity of patient sera for LJP 394 in-the second study. Thedata are mean±SEM for all patients in each group. The graph compares theKd′ before (solid bars) and after (slashed bars) 4 months of treatmentwith either 100 mg LJP 394 i.v. or placebo weekly.

FIG. 8 is a graph which depicts the patient population from FIG. 7divided into two groups, low affinity group (greater than Kd′ of 0.8IgG/ml serum) and high affinity group (less than Kd′ of 0.8 IgG/mlserum). Data are mean±SEM for all patients in each group. The graphcompares the Kd′ before (solid bars) and after (slashed bars) 4 monthsof treatment with either 100 mg LJP 394 i.v. or placebo weekly.

FIG. 9 is a graph which depicts the change in affinity of patient serafor LJP 394 epitope over time. The affinities are determined once amonth for the induction period of the trial (first 4 months of dosing at100 mg/wk). The x-axis is time elapsed in weeks; y-axis is Kd′. Thesamples were selected based on the initial (pre-treatment affinity) andsegregated into three groups of relative affinity: high, medium, and lowaffinities. The affinity values are the mean of 3 patients. The solidtriangles represent “low affinity patients” (Kd′ mean about 1.13 mg/ml);the solid circles represent “medium affinity” patients (Kd′ mean about0.44 mg/ml); and the solid squares represent “high affinity” patients(Kd′ mean about 0.14 mg/ml).

FIG. 10 is a graph which depicts the percent change in anti-ds DNAantibodies over time for high and low affinity patient groups treatedwith LJP 394, as assessed by a Farr assay. Data collected after highdose corticosteroid and/or cyclophosphamide treatment was excluded. Thepatients were stratified into low affinity (Kd′>0.8 mg/ml) and highaffinity (Kd′≦0.8 mg/ml) subgroups. The low affinity patients areindicated by the dotted line while the high affinity patients areindicated by the solid line. The data are means of all patients studied.The x-axis is time from start of trial in weeks and the y-axis is thepercent change in anti-dsDNA antibody levels from the start of thetrial. The gray regions mark the dosing periods, the first period is theinduction period with 100 mg/wk LJP 394 (or placebo) and the seconddosing period is with 50 mg/wk LJP 394 (or placebo). There is an “offperiod”, indicated by the white area of the graph, where no LJP 394 (orplacebo) is administered.

FIG. 11A is the same graph as FIG. 5A, except that incidence of activerenal flares in a patient are indicated by solid squares. FIG. 11B (LJP394-treated patients) is similar to FIG. 11A except that an expandedpopulation of patients has been used for analysis. FIG. 11C depicts thepercent change in Kd′ for placebo-treated patients using an expandedpopulation of patients. The patients experiencing flares are representedby solid squares for LJP 394-treated patients and solid circles forplacebo-treated patients.

FIG. 12 is a graph depicting renal flares in patients receivingconjugate LJP 394 (solid squares) and in patients receiving placebo(open squares). Each square represents a patient. X-axis is the K_(D)′before receiving treatment (or placebo); Y-axis is the percentage changein K_(D)′ after 4 months of treatment (or placebo).

FIG. 13 is a graph depicting change in K_(D)′ in patients receiving aplacebo (i.e., not receiving treatment) after four months, with solidsquares indicating incidence of renal flares and open squares indicatingplacebo patients. The results indicate a random distribution of flaresrelative to initial K_(D)′ and relative to change in K_(D′.)

FIG. 14 is a graph depicting time to the development of renal flares forthe high affinity patients. The solid line represents patients who havebeen treated with LJP 394 and the dotted line represents patients whohave been treated with a placebo. The x-axis is the time since firstdose of LJP 394 (or placebo) in months and the y-axis is the percentageof individuals that have not experienced a renal flare.

FIG. 15 is a graph depicting time to institution of high dosecorticosteroids and/or cyclophosphamide (HDCC) treatment in the highaffinity group. HDCC treatment is the first intervention with high dosecorticosteroid and/or cyclophosphamide in the high affinity patients.The solid line represents patients treated with LJP 394 and the dottedline represents patients treated with placebo. The x-axis is the timesince first dose of LJP 394 (or placebo) in months and the y-axis is thepercentage of individuals that have not received HDCC intervention.

FIG. 16 is a graph depicting a summary of clinical outcomes in the highaffinity group (Kd′≦0.8 mg/ml)).

FIG. 17 is a graph depicting a summary of clinical outcomes in theentire patient population (the “intent to treat” population).

MODES FOR CARRYING OUT THE INVENTION

We have discovered that average affinity of anti-dsDNA antibody from anindividual suffering from lupus to an epitope that will be or is used asa basis for treatment is predictive of efficacy of treatment designed toinduce immunotolerance by administering a conjugate containing at leasttwo molecules comprising the epitope. Based on our evaluation ofclinical data pertaining to such treatment in lupus patients, we havediscovered the following: (a) initial antibody affinity for the DNAepitope used in treatment can predict the degree of responsiveness totreatment, in terms of reduction of antibody affinity as well asreduction of symptoms and reduction of amount (titer) of anti-ds DNAantibody, with patients with higher affinity antibodies being betterresponders to treatment; (b) initial antibody affinity for the DNAepitope used in treatment can predict the degree of efficacy, in termsof reduction of symptoms, with patients with higher affinity antibodiesdisplaying reduction of symptoms compared to patients with loweraffinity antibodies (or patients with high affinity antibodies receivingno treatment); (c) change in antibody affinity for the DNA epitope usedin the treatment can predict the degree of efficacy in terms ofreduction of symptoms, with patients displaying a requisite change(reduction) of affinity displaying reduction of symptoms compared topatients failing to display the requisite change. In contrast, previousreports disclosed measuring titer of anti-ds DNA antibodies uponadministration of a conjugate described herein (LJP 394) (i.e., levelsof anti-ds DNA antibodies as indicated by binding to ds DNA), as opposedto reporting affinity of a patient's anti-ds DNA antibodies for thepolynucleotide epitope of the conjugate. In view of our analysis,neither these initial reported titers or changes in titers werepredictive of clinical outcome.

Accordingly, the invention provides methods of treatment and methods ofidentifying individuals suitable for the treatments described hereinwhich entail assessment of affinity of antibody from an individual foran epitope that is, has been, and/or will be, the basis ofimmunotolerance treatment as described herein.

General Techniques

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature, such as, Molecular Cloning: ALaboratory Manual, second edition (Sambrook et al., 1989) Cold SpringHarbor Press; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); AnimalCell Culture (R. I. Freshney), ed., 1987); Methods in Enzymology(Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir& C. C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller & M. P. Calos, eds., 1987); Current Protocols in MolecularBiology (F. M. Ausubel et al., eds., 1987); PCR: The Polymerase ChainReaction, (Mullis et al., eds., 1994); Current Protocols in Immunology(J. E. Coligan et al., eds., 1991) and Short-Protocols in MolecularBiology (Wiley and Sons, 1999).

DEFINITIONS

“Affinity” of an antibody from an individual for an epitope to be used,or used, in treatment(s) described herein is a term well understood inthe art and means the extent, or strength, of binding of antibody toepitope. Affinity may be measured and/or expressed in a number of waysknown in the art, including, but not limited to, equilibriumdissociation constant (K_(D)or K_(d)), apparent equilibrium dissociationconstant (K_(D)′ or K_(d)′), and IC₅₀ (amount needed to effect 50%inhibition in a competition assay; used interchangeably herein with“I₅₀”). It is understood that, for purposes of this invention, anaffinity is an average affinity for a given population of antibodieswhich bind to an epitope. Values of K_(D)′ reported herein in terms ofmg IgG per ml or mg/ml indicate mg Ig per ml of serum, although plasmacan be used.

When antibody affinity “is used as a basis” for administration of thetreatment methods described herein, or selection for the treatmentmethods described herein, antibody affinity is measured before and/orduring treatment, and the values obtained are used by a clinician inassessing any of the following: (a) probable or likely suitability of anindividual to initially receive treatment(s); (b) probable or likelyunsuitability of an individual to initially receive treatment(s); (c)responsiveness to treatment; (d) probable or likely suitability of anindividual to continue to receive treatment(s); (e) probable or likelyunsuitability of an individual to continue to receive treatment(s); (f)adjusting dosage; (g) predicting likelihood of clinical benefits. Aswould be well understood by one in the art, measurement of antibodyaffinity in a clinical setting is a clear indication that this parameterwas used as a basis for initiating, continuing, adjusting and/or ceasingadministration of the treatments described herein.

An antibody affinity measured “before or upon initiation of treatment”is antibody affinity measured in an individual before the individualreceives the first administration of a treatment modality describedherein and/or within at least about 4 weeks, preferably within at leastabout 2 weeks, preferably within at least about 1 week, preferablywithin at least about 5 days, preferably within at least about 3 days,preferably within at least about 2 days, preferably within at leastabout 1 day upon receiving the first administration of a treatmentmodality described herein.

A “population” is a group of individuals with an antibody-mediatedpathology. For a given population (which may vary in terms of number ofmembers, depending on the context) antibody affinities vary over a range(i.e., maximum and minimum affinities).

An individual who “may be suitable”, which includes an individual who is“suitable” for treatment(s) described herein, is an individual who ismore likely than not to benefit from administration of said treatments.Conversely, an individual who “may not be suitable” or “may beunsuitable”, which includes an individual who is “unsuitable” fortreatment(s) described herein, is an individual who is more likely thannot to fail to benefit from administration of said treatments.

As used herein, “treatment” is an approach for obtaining beneficial ordesired results including and preferably clinical results. For purposesof this invention, beneficial or desired clinical results include, butare not limited to, one or more of the following: alleviation ofsymptoms, diminishment of extent of disease, stabilized (i.e., notworsening) state of disease, preventing spread (i.e., metastasis) ofdisease, preventing occurrence or recurrence of disease, delay orslowing of disease progression, amelioration of the disease state,remission (whether partial or total), reduction of incidence of diseaseand/or symptoms. Treatment of lupus includes any aspect of lupus,including, but not limited to, lupus nephritis, which is a chronicinflammatory kidney disease. During lupus nephritis, “flares” may occur.“Flares” refer to an increase in activity, generally inflammatoryactivity. If the activity is in the kidneys, then the flare is referredto as a “renal flare”. “Renal flares” can be identified by evaluatingfactors including, but not limited to, proteinuria levels, hematurialevels, and serum creatinine levels. The “treatment” of lupus nephritismay be administered when no symptoms of lupus nephritis are present, andsuch treatment (as the definition of “treatment” indicates) reduces theincidence of flares. Also encompassed by “treatment” is a reduction ofpathological consequences of any aspect of lupus, such as lupusnephritis.

“SLE flares” are used herein to refer to flares (i.e. acute clinicalevents) which occur in patients with SLE. The SLE flares may be invarious major organs, including but not limited to, kidney, brain, lung,heart, liver, and skin. SLE flares include renal flares.

“High dose corticosteroid and/or cyclophosphamide” or “HDCC” as usedherein refers to intervention with an increased dosage of corticosteroidalone or with cyclophosphamide. High dose generally refers tocorticosteroids. Such intervention generally occurs upon a flare, oracute episode. Generally, for example, the increase dosage is at least a15 mg/day and can be greater than 20 mg/day. HDCC may be administeredusing standard clinical protocols. A clinician may monitor a patient anddetermine when HDCC treatment is needed by evaluating factors including,but not limited to, proteinuria levels, hematuria levels, and serumcreatinine levels. In general, patients who experience renal flares aregiven HDCC treatment, although this treatment is used for other aspectsof lupus.

An “equivalent” or “functional equivalent” of K_(D)′ or a numericalvalue for K_(D)′ is a parameter or value for a parameter which alsoreflects affinity. For example, an equivalent of K_(D)′ is IC₅₀. Asanother example, an equivalent value of K_(D)′ of 0.5 could be an IC₅₀of 200, if they reflect the same, or about the same, affinity.Determining such equivalents is well within the skill of the art andsuch equivalents and their determination are encompassed by thisinvention. Generally, reference to K_(D)′ includes reference tofunctional equivalents of K_(D′.)

As used herein, the singular form “a”, “an”, and “the” includes pluralreferences unless indicated otherwise. For example, “an” antibodyincludes one or more antibodies.

An “epitope” is a term well-understood in the art and means any chemicalmoiety which exhibits specific binding to an antibody. An “epitope” canalso comprise an antigen, which is a moiety or molecule that contains anepitope, and, as such, also specifically binds to antibody.

A “double-stranded DNA epitope” or “dsDNA epitope” is any chemicalmoiety which exhibits specific binding to an anti-double-stranded DNAantibody and as such includes molecules which comprise such epitope(s).Further discussion of double-stranded DNA epitopes suitable for theconjugates of the invention are described below. The term “epitope” alsoincludes mimetics of double-stranded DNA itself, which are describedbelow.

An epitope that “specifically binds” to an antibody is a term wellunderstood in the art, and methods to determine such specific bindingare also well known in the art. A molecule is said to exhibit “specificbinding” if it reacts or associates more frequently, more rapidly, withgreater duration and/or with greater affinity with a particular cell orsubstance than it does with alternative cells or substances. An antibody“specifically binds” to a target if it binds with greater affinity,avidity, more readily, and/or with greater duration than it binds toother substances.

An “anti-double-stranded DNA antibody” or “anti-dsDNA antibody” or“double-stranded DNA antibody”, used interchangeably herein, is anyantibody which specifically binds to double-stranded DNA (dsDNA). Anyantibody includes an antibody of any class, such as IgG, IgA, or IgM,and the antibody need not be of any particular class. As clearlyindicated in the definition of “antibody” provided herein, a“anti-double-stranded DNA antibody” encompasses any fragment(s) thatexhibits this requisite functional (i.e., specific binding to dsDNA)property, such as fragments that contain the variable region, such asFab fragments. As discussed below, it is understood that specificbinding to any anti-double-stranded DNA antibody (or functionalfragment) is sufficient.

The term “circulating anti-double-stranded DNA antibody”, as usedherein, intends an anti-double-stranded DNA antibody which is not boundto a double-stranded DNA epitope on and/or in a biological sample, i.e.,free antibody.

An “antibody” (interchangeably used in plural form) is an immunoglobulinmolecule capable of specific binding to a target, such as acarbohydrate, polynucleotide or polypeptide, through at least oneantigen recognition site, located in the variable region of theimmunoglobulin molecule. As used herein, the term encompasses not onlyintact antibodies, but also fragments thereof (such as Fab, Fab′,F(ab′)₂, Fv), single chain (ScFv), mutants thereof, fusion proteinscomprising an antibody portion, humanized antibodies, and any othermodified configuration of the immunoglobulin molecule that comprises anantigen recognition site of the required specificity.

The terms “polynucleotide” and “nucleic acid”, used interchangeablyherein, refer to a polymeric form of nucleotides of any length, eitherribonucleotides or deoxyribonucleotides. These terms include a single-,double- or triple-stranded DNA, genomic DNA, cDNA, RNA, DNA-RNA hybrid,or a polymer comprising purine and pyrimidine bases, or other natural,chemically, biochemically modified, non-natural or derivatizednucleotide bases. For purposes of this invention, unless otherwiseindicated, sequences presented herein denote double stranded sequences.For example, the polynucleotide comprising, consisting essentially of,or consisting of the double stranded sequence 5′-GTGTGTGTGTGTGTGTGTGT-3′includes the complementary polynucleotide sequence, particularly thesequence 3′-CACACACACACACACACACA-5′. It is understood that the doublestranded polynucleotide sequences described herein also include themodifications described herein. The backbone of the polynucleotide cancomprise sugars and phosphate groups (as may typically be found in RNAor DNA), or modified or substituted sugar or phosphate groups.Alternatively, the backbone of the polynucleotide can comprise a polymerof synthetic subunits such as phosphoramidates and thus can be aoligodeoxynucleoside phosphoramidate (P-NH2) or a mixedphosphoramidate-phosphodiester oligomer. A phosphorothioate linkage canbe used in place of a phosphodiester linkage. In addition, adouble-stranded polynucleotide can be obtained from the single strandedpolynucleotide product of chemical synthesis either by synthesizing thecomplementary strand and annealing the strands under appropriateconditions, or by synthesizing the complementary strand de novo using aDNA polymerase with an appropriate primer.

The following are non-limiting examples of polynucleotides: a gene orgene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA,recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,nucleic acid probes, and primers. For purposes of this invention, apolynucleotide is generally an isolated polynucleotide of less thanabout 1 kb, preferably less than about 500 base pairs (bp), preferablyless than about 250 bp, preferably less than about 100 bp, preferablyless than about 50 bp. However, it is understood that a polynucleotideof any size or configuration could be used as long as it exhibits therequisite binding to anti ds DNA antibody from an individual. It isfurther understood that a different polynucleotide (for example, interms of size and/or sequence) other than the one that is to be, was, orwill be used in treatment, as long as both polynucleotides exhibitequivalent (or convertible) binding affinities to anti-ds DNA antibodiesfrom an individual. In other words, non-identical polynucleotides may beemployed with respect to affinity determination and treatment.

Preferably, the polynucleotide is DNA. As used herein, “DNA” includesnot only bases A, T, C, and G, but also includes any of their analogs ormodified forms of these bases, such as methylated nucleotides,internucleotide modifications such as uncharged linkages and thioates,use of sugar analogs, and modified and/or alternative backbonestructures, such as polyamides.

“Naturally occurring” refers to an endogenous chemical moiety, such as acarbohydrate, polynucleotide or polypeptide sequence, i.e., one found innature. Processing of naturally occurring moieties can occur in one ormore steps, and these terms encompass all stages of processing.Conversely, a “non-naturally occurring” moiety refers to all othermoieties, i.e., ones which do not occur in nature, such as recombinantpolynucleotide sequences and non-naturally occurring carbohydrates.

As used herein, the term “immunogen” means a chemical entity thatelicits a humoral immune response when injected into an animal.Immunogen have both B cell epitopes and T cell epitopes.

As used herein, the term “analog” (also termed an “mimetic”) of animmunogen means a biological or chemical compound which specificallybinds to an antibody to which the immunogen specifically binds. As sucha “double-stranded DNA epitope” includes mimetics of naturally-occurringdouble-stranded DNA. An “analog” or “mimetic” shares an epitope, orbinding specificity, with double-stranded DNA. An analog may be anychemical substance which exhibits the requisite binding properties, andthus may be, for example, a simple or complex organic or inorganicmolecule; a polypeptide; a polynucleotide; a carbohydrate; a lipid; alipopolysaccharide; a lipoprotein, or any combination of the above,including, but not limited to, a polynucleotide-containing polypeptide;a glycosylated polypeptide; and a glycolipid. The term “analog”encompasses the term “mimotope”, which is a term well known in the art.

An “individual” is a vertebrate, preferably a mammal, more preferably ahuman. Mammals include, but are not limited to, farm animals, sportanimals, pets, primates, mice and rats.

A “T cell epitope” means a component or portion thereof for which a Tcell has an antigen-specific specific binding site, the result ofbinding to which activates the T cell. Where an embodiment of theinvention is described as “lacking” a T cell epitope, this is taken tomean that a T cell epitope is not detectable using standard assays inthe art. For purposes of this invention, an epitope that “lacks” a Tcell epitope means that the epitope lacks a T cell epitope which causesT cell activation in the individual(s) to be treated (i.e., who is toreceive an epitope-presenting valency platform molecule). It is likelythat, for example, an epitope may lack a T cell epitope(s) with respectto an individual, or a group of individuals, while possessing a T cellepitope(s) with respect to other individual(s). Methods for detectingthe presence of a T cell epitope are known in the art and include assayswhich detect T cell proliferation (such as thymidine incorporation).Immunogens that fail to induce statistically significant incorporationof thymidine above background (i.e., generally p less than 0.05 usingstandard statistically methods) are generally considered to lack T cellepitopes, although it will be appreciated that the quantitative amountof thymidine incorporation may vary, depending on the immunogen beingtested. Generally, a stimulation index below about 2-3, more preferablyless than about 1, indicates lack of T cell epitopes. The presence of Tcell epitopes can also be determined by measuring secretion of Tcell-derived lymphokines according to standard methods. Location andcontent of T cell epitopes, if present, can be determined empirically.It is understood that, over time, more sensitive assays may be developedto detect the presence of T cell epitopes, and that specifying the lackof T cell epitopes is dependent on the type of detection system used.

“Inducing tolerance” or “inducing immunotolerance” means a reductionand/or stabilization of the extent of an immune response to animmunogen. An “immune response” may be humoral and/or cellular, and maybe measured using standard assays known in the art. For purposes of thisinvention, the immune response is generally reflected by the presenceof, and/or the levels of, anti-double-stranded DNA antibodies.Quantitatively the reduction (as measured by reduction in antibodyproduction and/or levels) is at least about: 15%, preferably at leastabout 25%, more preferably at least about 50%, more preferably at leastabout 75%, more preferably at least about 90%, even more preferably atleast about 95%, and most preferably 100%. It is understood that thetolerance is antigen-specific, and applies for purposes of the inventionto those individuals having anti-double-stranded DNA antibodies.“Inducing tolerance” also includes slowing and/or delaying the rate ofincrease of antibody level.

As used herein, the term “B cell anergy” intends unresponsiveness ofthose B cells requiring T cell help to produce and secrete antibody andincludes, without limitation, clonal deletion of immature and/or matureB cells and/or the inability of B cells to produce antibody.“Unresponsiveness” means a therapeutically effective reduction in thehumoral response to an immunogen. Quantitatively the reduction (asmeasured by reduction in antibody production) is at least 50%,preferably at least 75% and most preferably 100%.

An “effective amount” (when used in the lupus context, or in theantibody-mediated pathology context) is an amount sufficient to effectbeneficial or desired results including clinical results. An effectiveamount can be administered in one or more administrations. For purposesof this invention, an effective amount of conjugate described herein (ora composition comprising a conjugate) an amount sufficient to reducecirculating levels of anti-double-stranded DNA antibodies, preferably byinducing tolerance, particularly with respect to anti-double-strandedDNA antibodies. In terms of treatment, an “effective amount” ofconjugate described herein (or a composition comprising a conjugate) isan amount sufficient to palliate, ameliorate, stabilize, reverse, slowor delay progression of or prevent systemic lupus erythematosus (SLE),including the progressive inflammatory degeneration of the kidneys thatresults from SLE (i.e., lupus nephritis).

A “stable complex” formed between any two or more components in abiochemical reaction, refers to a duplex or complex that is sufficientlylong-lasting to persist between formation of the duplex or complex andsubsequent detection, including any optional washing steps or othermanipulation that may take place in the interim.

An “isolated” or “purified ” polypeptide or polynucleotide is one thatis substantially free of the materials with which it is associated innature. By substantially free is meant at least 50%, preferably at least70%, more preferably at least 80%, even more preferably at least 90%free of the materials with which it is associated in nature.

As used herein “valency platform molecule” means a nonimmunogenicmolecule containing sites which allow the attachment of a discretenumber of epitopes and/or mimetic(s) of epitopes. A “valency” of aconjugate or valency platform molecule indicates the number ofattachment sites per molecule for a double-stranded DNA epitope(s).Alternatively, the valency of a conjugate is the ratio (whether absoluteor average) of double-stranded DNA epitope to valency platform molecule.

“Nonimmunogenic”, when used to describe the valency platform molecule,means that the valency platform molecule fails to elicit an immuneresponse (i.e., T cell and/or B cell response), and/or fails to elicit asufficient immune response, when it is administered by itself to anindividual. The degree of acceptable immune response depends on thecontext in which the valency platform molecule is used, and may beempirically determined.

An epitope which is “conjugated” to a valency platform molecule is onethat is attached to the valency platform molecule, either by covalentand/or non-covalent interactions.

An “epitope-presenting valency platform molecule” is a valency platformmolecule which contains attached, or bound, epitopes, at least some ofwhich (at least two of which) are able to bind an antibody of interest.

A “biological sample” encompasses a variety of sample types obtainedfrom an individual and can be used in a diagnostic or monitoring assay.The definition encompasses blood and other liquid samples of biologicalorigin, solid tissue samples such as a biopsy specimen or tissuecultures or cells derived therefrom, and the progeny thereof. Thedefinition also includes samples that have been manipulated in any wayafter their procurement, such as by treatment with reagents,solubilization, or enrichment for certain components, such as proteinsor polynucleotides. The term “biological sample” encompasses a clinicalsample, and also includes cells in culture, cell supernatants, celllysates, serum, plasma, biological fluid, and tissue samples.

“In conjunction with” refers to administration of one treatment modalityin addition to another treatment modality, such as administration of aconjugate described herein in addition to administration ofcorticosteroid cyclophosphamide immunosuppressants (or otherimmunosuppressant therapy) to the same individual. As such, “inconjunction with” refers to administration of one treatment modalitybefore, during or after delivery of the other treatment modality to theindividual.

An “antibody-mediated pathology” is an immune response disorder which isassociated with inappropriate production of antibodies; generallydirected to self-antigens. Antibody-mediated pathologies include, butare not limited to, lupus; antibody-mediated thrombosis andthrombocytopenia; anti-phospholipid syndrome; myasthenia gravis. Becausean immune response disorder is context dependent, for purposes of thisinvention, an “antibody-mediated pathology” can also encompasstransplantation rejection (especially xenotransplantation), in which animmune response is inappropriate with respect to attempting to maintainthe foreign transplanted tissue, and Rh-based rejection in pregnancy.

“Receiving treatment” includes initial treatment and/or continuingtreatment.

“Comprising” means including.

Methods of Treatment of Lupus and Methods of Identifying IndividualsSuitable for Treatment of Lupus Based on Assessment of Antibody Affinity

The methods described herein entail assessing antibody affinity from anindividual, wherein said individual has, or is suspected of having,systemic lupus erythematosus (SLE). For purposes of this invention, (a)the affinity in question is with respect to an individual's antibodies,that is, antibodies obtained from that individual; (b) the antibody forwhich affinity is measured is an antibody associated with, and/orimplicated in, an antibody-mediated pathology, namely systemic lupuserythematosus (SLE); and (c) the binding of interest is binding ofantibody to an epitope which binds to the antibody(ies), with theepitope to be used in the proposed treatment, as described herein (i.e.,a dsDNA epitope).

The invention provides (a) methods of treating individuals based onassessment of initial antibody affinity (i.e., antibody affinity beforeor upon initiation of treatment); (b) methods of treating individualsbased on assessment of change in antibody affinity, if any (i.e.,comparison of (i) antibody affinity before or upon initiation oftreatment with (ii) antibody affinity after a period treatmentsufficient to elicit a change in affinity, if any); (c) methods ofscreening individuals who may be suitable for receiving the treatment(s)described herein (or, alternatively, individuals who may be unsuitableto receive the treatment(s) described herein) based on assessment ofinitial antibody affinity; (d) methods of screening individuals who maybe suitable for receiving treatment(s) described herein (or,alternatively, individuals who may be unsuitable to receive treatment(s)described herein) based on assessment of the change in antibodyaffinity, if any; (e) methods of monitoring the treatment(s) describedherein based on assessment of antibody affinity.

With respect to the methods described herein, the screening methods(i.e., methods of identifying individuals as suitable or unsuitable fortreatment) may be practiced independently of the treatment methods, andas such are distinct from treatment methods. The screening methodsdescribed herein may be practiced by a skilled technician other than amedical doctor, using equipment and/or techniques of the art.

For all embodiments of the invention which use or are directed toK_(D)′, whether screening, treatment, monitoring, or any other methodsdirected to assessing affinity, it is understood that other, equivalentvalues can be measured and used, and are encompassed by this invention.For example, as discussed below, there are a number of methods known inthe art which can measure (and express) affinity of antibodies from anindividual for an epitope to be used for treatment (in the context ofthis invention, a double stranded DNA epitope). K_(D)′ is one of theseparameters, and equivalent parameters can be measured and used in thisinvention. Further, with respect to K_(D)′ cut-off values reportedherein, the basis of this finding was administering about 100 mg of LJP394 conjugate about once a week.

Methods of Treatment

Accordingly, the invention provides methods of treating SLE in anindividual, comprising administering to the individual a conjugatecomprising (a) a non-immunogenic valency platform molecule and (b) twoor more dsDNA epitopes (such as two or more molecules comprising a dsDNAepitope), preferably polynucleotides, which specifically bind to anantibody from the individual which specifically binds to double strandedDNA, wherein affinity of the epitope, preferably polynucleotide (of theconjugate) for the antibody from the individual is used as a basis forselecting the individual to receive the treatment. In other embodiments,the invention provides methods of treating SLE in an individual,comprising administering to the individual a conjugate comprising (a) anon-immunogenic valency platform molecule and (b) two or more dsDNAepitopes, preferably polynucleotides, which specifically bind to anantibody from the individual which specifically binds to double strandedDNA, wherein affinity of the epitope, preferably polynucleotide (of theconjugate) for the antibody from the individual is used as a basis forselecting the individual to continue to receive the treatment. Methodsof determining affinities are known in the art and described below.

Measurement of affinity, either represented by measuring K_(D)′ or bysome other method, either before or during treatment is strong, if notconclusive, indication that this parameter was a basis for selecting theindividual to receive (and/or continue to receive) treatment.Accordingly, with respect to all treatment methods described herein, andas the definition for “is used as a basis” states, other embodimentsinclude (1) assessing, or measuring, the affinity as described herein(and preferably selecting an individual suitable for receiving(including continuing to receive) treatment); and (2) administering thetreatment(s) as described herein. As described herein, in someembodiments, more than one measurement is made, when change (if any) inaffinity is assessed.

dsDNA epitopes for use in the treatment methods are described herein. Insome embodiments, the ds DNA epitope is a polynucleotide, such as5′-GTGTGTGTGTGTGTGTGTGT-3′. Affinity may be measured using the epitope(or a molecule or moiety comprising the epitope) used in the conjugate;alternatively, a similar, non-identical epitope may be used, as long asits affinity may be at least correlated to the affinity of the epitopeused in the conjugate, so that a meaningful measurement of affinity maybe obtained.

The invention provides methods of treating SLE in an individual,comprising administering to the individual a conjugate comprising (a) anon-immunogenic valency platform molecule and (b) two or morepolynucleotides which specifically bind to an antibody from theindividual which specifically binds to double stranded DNA, saidpolynucleotide comprising, consisting essentially of, or consisting ofthe double stranded sequence 5′-GTGTGTGTGTGTGTGTGTGT-3′, wherein theapparent equilibrium dissociation constant (K_(D)′) for thepolynucleotide in the conjugate with respect to the antibody from theindividual before or upon initiation of treatment is less than about 1.0mg IgG per ml, and wherein said K_(D)′ value is used as a basis forselecting the individual to receive the treatment. In other embodiments,the K_(D)′ is less than about any of the following: 0.8; 0.7; 0.6; 0.5;0.4; 0.3; 0.2; 0.1; 0.09; 0.08; 0.07; 0.06; 0.05; 0.025. These valuesfor K_(D)′ apply to all methods in which K_(D)′ is assessed (includingtreatment and/or screening), such as those in which treatment based on5′-GTGTGTGTGTGTGTGTGTGT-3′ is contemplated. In some embodiments, K_(D)′is less than about 0.8 mg IgG per ml. In some embodiments, K_(D)′ isless than about 0.5 mg IgG per ml. In some embodiments, K_(D)′ is lessthan about 0.1 mg IgG per ml. Methods of measuring K_(D)′ are describedbelow. Measurement of affinity, either represented by measuring K_(D)′or by some other method, either before or during treatment is strong, ifnot conclusive, indication that this parameter was a basis for selectingthe individual to receive treatment.

In some embodiments, the invention provides methods of treating SLE inan individual, comprising: (a) assessing before or upon initiation oftreatment an apparent equilibrium dissociation constant (K_(D)′) for adsDNA epitope (including a molecule comprising a dsDNA epitope),preferably a polynucleotide in or of a conjugate with respect toantibodies from the individual which specifically bind to doublestranded DNA, said conjugate comprising (a) a non-immunogenic valencyplatform molecule and (b) two or more DNA epitopes, preferablypolynucleotides, which specifically bind to an antibody from theindividual which specifically binds to double stranded DNA, saidpolynucleotide (if that is the dsDNA epitope used) preferablycomprising, consisting essentially of or consisting of the (doublestranded, or ds) sequence 5′-GTGTGTGTGTGTGTGTGTGT-3′, wherein theindividual is selected to receive the treatment if the K_(D)′ is lessthan about 1.0 mg IgG per ml; and (b) administering to the individualthe conjugate, preferably in an amount sufficient to increase theK_(D)′. In other embodiments, the K_(D)′ is less than about any of thefollowing: 0.8; 0.7; 0.6; 0.5; 0.4; 0.3; 0.2; 0.1; 0.09; 0.08; 0.07;0.06; 0.05.; 0.025. Methods of measuring K_(D)′ are described below.

In other embodiments, the invention provides methods of treating SLE inan individual, comprising: (a) assessing before or upon initiation oftreatment an apparent equilibrium dissociation constant (K_(D)′) for adsDNA epitope, preferably a polynucleotide in or of a conjugate withrespect to antibodies from the individual which specifically bind todouble stranded DNA, said conjugate comprising (a) a non-immunogenicvalency platform molecule and (b) two or more such epitopes, preferablypolynucleotides, which specifically bind to an antibody from theindividual which specifically binds to double stranded DNA, saidpolynucleotide (if used) comprising, consisting essentially of orconsisting of the (ds) sequence 5′-GTGTGTGTGTGTGTGTGTGT-3′; and (b)administering to the individual the conjugate in an amount sufficient toincrease the K_(D)′, wherein treatment is continued if K_(D)′ isincreased at least about 20% compared to K_(D)′ before or uponinitiation of treatment. For these embodiments, a K_(D)′ measured afterinitiation of treatment (for comparison to K_(D)′ before or uponinitiation of treatment) is measured at least about 4 weeks, preferablyat least about 6 weeks, more preferably at least about 10 weeks, morepreferably at least about 12 weeks, after initiation of treatment. Weobserved a large range of change in antibody affinity upon treatmentover a treatment population (FIG. 5). Accordingly, in other embodiments,treatment is continued if K_(D)′ is increased at least about any of thefollowing (as compared to K_(D)′ before or upon initiation oftreatment): 40%,50%,75%,100%, 200%, 500%. Methods of measuring K_(D)′are described below.

In some embodiments, a conjugate is administered in an amount sufficientto reduce incidence of, or likelihood of, renal flares (lupusnephritis). Based on our observations, the amount sufficient for thisreduction for LJP 394 is about 100 mg, given once per week. Patientsreceiving this amount of conjugate, who had an initial K_(D)′ of lessthan about 0.8, displayed more than about two-fold lowerhospitalizations due to this disorder. For an individual patient, aneffective amount of conjugate may be related to and/or a function of,that individual's antibody affinity, with patients with higher affinityable to respond to a lower dose. Conversely, a patient with loweraffinity antibodies may respond if given a sufficiently high(er) dose.

Accordingly, the invention provides methods of treating lupus nephritisin an individual, comprising administering to the individual a conjugatecomprising (a) a non-immunogenic valency platform molecule and (b) twoor more polynucleotides which specifically bind to an antibody from theindividual which specifically binds to double stranded DNA, saidpolynucleotide comprising, consisting essentially of, or consisting ofthe (ds) sequence 5′-GTGTGTGTGTGTGTGTGTGT-3′, wherein the apparentequilibrium dissociation constant (K_(D)′) for the polynucleotide in theconjugate with respect to the antibody from the individual before orupon initiation of treatment is less than about 1.0 mg IgG per ml, andwherein said K_(D)′ value is used as a basis for selecting theindividual to receive the treatment. In other embodiments, the K_(D)′ isless than about any of the following: 0.8; 0.7; 0.6; 0.5; 0.4;,0.3; 0.2;0.1; 0.09; 0.08; 0.07; 0.06 0.05; 0.025. Methods of measuring K_(D)′ aredescribed below. Measurement of affinity, either represented bymeasuring K_(D)′ or by some other method, either before or duringtreatment is strong, if not conclusive, indication that this parameterwas a basis for selecting the individual to receive treatment.

In some embodiments, a conjugate as described herein is administered inan amount sufficient to reduce the dosage of corticosteroid and/orcyclophosphamide immunosuppressive therapy that would otherwise beadministered in the absence of administering the conjugate. This issignificant, as this type of immunotherapy is toxic. Accordingly, theinvention provides methods of treating lupus nephritis wherein aconjugate as described herein (such as LJP 394) is administered in anamount sufficient to reduce the amount of corticosteroid orcyclophosphamide administered to the individual as compared to usingcorticosteroid or cyclophosphamide without administering the conjugate.In some embodiments, the incidence of renal flares are reduced in theindividual and the conjugate is administered in an amount sufficient toeffect this reduction. The invention also provides methods of treatingSLE, preferably lupus nephritis, comprising administering a (any)conjugate described herein in conjunction with corticosteroid and/orcyclophosphamide. The conjugate is administered in an amount effectiveto reduce antibody affinity for the epitope in the conjugate.Preferably, the conjugate is LJP 394. Methods of administeringcorticosteroid and/or cyclophosphamide are known in the art. Reducingthe dosage of corticosteroid and/or cyclophosphamide therapy (whichreduces the dependence on administration of these drugs and in effectdelays administration of these drugs) can be assessed by, for example,comparing to known and/or established averages of dosage (in terms ofamount and/or intervals) generally given over time which are known inthe art.

Methods of Screening

The invention also provides methods of identifying individuals who maybe suitable to receive (and/or to continue to receive) the treatmentsdescribed herein, or, in alternative embodiments, methods of identifyingindividuals who may be unsuitable to receive (and/or to continue toreceive) the treatments described herein, based on antibody affinities.

Accordingly, in some embodiments, the invention provides methods ofidentifying an individual who may be suitable for treatment for SLE,said treatment comprising administration of a conjugate comprising (a) anon-immunogenic valency platform molecule and (b) two or more dsDNAepitopes, preferably polynucleotides which specifically bind to anantibody from the individual which specifically binds to double strandedDNA, said polynucleotide (if a polynucleotide is used) comprising,consisting essentially of or consisting of the (ds) sequence5′-GTGTGTGTGTGTGTGTGTGT-3′, said method comprising measuring theapparent equilibrium dissociation constant (K_(D)′) for thepolynucleotide in (or of) conjugate before or upon initiation oftreatment and anti-double stranded DNA antibodies from the individual,wherein an individual is identified by K_(D)′ (or its functionalequivalent) of less than about 1.0 mg IgG per ml. In other embodiments,the K_(D)′ is less than about any of the following: 0.8; 0.7; 0.6; 0.5;0.4; 0.3; 0.2; 0.1; 0.09; 0.08; 0.07; 0.06 0.05; 0.025. The inventionthus provides screening based on any of a number of dsDNA epitopescontemplated for use in treatment. Generally, a higher affinity“cut-off” (for example, as indicated by a lower K_(D)′ value) wouldprovide a higher degree of certainty with respect to likely success oftreatment.

In other embodiments, the invention provides methods of identifying anindividual who may be unsuitable for treatment for SLE, said treatmentcomprising administration of a conjugate comprising (a) anon-immunogenic valency platform molecule and (b) two or more dsDNAepitopes, preferably polynucleotides which specifically bind to anantibody from the individual which specifically binds to double strandedDNA, said polynucleotide (if used) comprising, consisting essentially ofor consisting of the (ds) sequence 5′-GTGTGTGTGTGTGTGTGTGT-3′, saidmethod comprising measuring the apparent equilibrium dissociationconstant (K_(D)′) for the polynucleotide in (or of) conjugate andanti-double stranded DNA antibodies from the individual before or uponinitiation of treatment, wherein an individual is identified by K_(D)′of more than about 1.0 mg IgG per ml. In other embodiments, theindividual is identified by a K_(D)′ of more than about 0.8 mg IgG perml. If expressed as a range, the upper limit may be any number,including, but not limited to, about 2.0, about 3.0, about 5.0, about10, about 15, about 20.

The invention also provides methods of monitoring treatment for SLE inan individual, said treatment comprising administration of a conjugatecomprising (a) a non-immunogenic valency platform molecule and (b) twoor more dsDNA epitopes, preferably polynucleotides which specificallybind to an antibody from the individual which specifically binds todouble stranded DNA, said method comprising measuring the affinity (forexample, the apparent equilibrium dissociation constant (K_(D)′)) of theconjugate or dsDNA epitope (such as polynucleotide) and anti-doublestranded DNA antibodies from the individual. For these methods, a changein K_(D)′ (compared to initial K_(D)′) generally indicates that thetreatment may be continued (i.e., that the treatment will beefficacious). The change in K_(D)′ may be at least about any of thechanges described herein (generally from at least about 20% to at leastabout 500%), with measurement(s) generally, but not necessarily, atleast two to six weeks apart. A change over any two given measurements(whether or not sequential) may be used. For example, if measurement attwo weeks shows no change, but comparison of the measurement at fourweeks shows a change, continuation of treatment may be indicated.Treatment could also be continued if K_(D)′ does not change or evenchanges indicating an increase in affinity, as such developments(including no change) could be temporary. Alternatively, a change inaffinity (such as K_(D)′) need not be tested for or observed. Forexample, affinity could be measured and compared to that from a generalpopulation (e.g., an average affinity). Thus, with respect embodimentsdirected to methods of monitoring treatment, a particular result neednot be observed for these methods to be practiced, although certainresults (or ranges of results) may be desirable and can be used.

In some embodiments, an individual is selected for treatment based on apercentile ranking of affinity compared to a population. For example,there is a range of antibody affinities over a given patient population,and individuals suitable for treatment (or, conversely, individualslikely to be unsuitable) can be identified based on a percentile rankingof antibody affinity with respect to this population. Accordingly, insome embodiments, an individual is included in treatment, or identifiedas suitable to receive treatment, if the antibody affinity for thatindividual is in about the top 80% of affinities for that population(conversely, individuals are generally not suitable to receive treatmentif they are in about the bottom 20% of affinities for that population).In other embodiments, an individual is included in treatment, oridentified as suitable to receive treatment, if the antibody for thatindividual is in about the top 50% of affinities for that population(conversely, individuals are generally not suitable to receive treatmentif they are in about the bottom 50% of affinities for that population).In some embodiments, the antibody in that individual is in about any ofthe top percentages: 30%; 25%; 20%;. 10%; 5%. A population may be about,or alternatively at least about any of the following, in terms of numberof individuals measured: 10, 15, 20, 25, 30, 50, 60, 75, 100, 125, 150,175, 200, 225, 250, 300, 400, 500. Preferably, a sufficient number ofindividuals are measured to provide a statistically significantpopulation, which can be determined by methods known in the art. Anupper limit of a population may be any number, including those listed.

Generally, an individual is suitable to receive the treatments (or tocontinue to receive the treatments) described herein if, afteradministering the conjugate(s) in an amount sufficient and for a timesufficient to elicit a response in terms of reducing antibody affinity,the individual's antibody affinity decreases at least about any 20% withrespect to antibody affinity before administration of conjugate. Forexample, a 20% reduction in antibody affinity is reflected in a changeof an initial K_(D)′ of 0.4 to a K_(D)′ of 0.5. In other embodiments,the individual's antibody affinity decreases by at least about 50%. Inother embodiments, the individual's antibody affinity decreases by atleast about 75%. Based on our observations in the context of lupustreatments, at least about 8 weeks, preferably at least about 10 weeks,more preferably at least about 12 weeks, should be a sufficient time toobserve a measurable response. Lack of a measurable change in affinityover this time period indicates that the individual receiving thetreatment is unlikely to benefit from such treatment, in terms ofreduction of symptoms.

Generally, a response is indicated (and therefore continuation oftreatment is indicated) if there is a statistically significant change(reduction) compared to the initial affinity (i.e., affinity measuredbefore or upon initiation of treatment). A statistically significantchange depends upon the particular assay used, and this value isgenerally readily obtainable. For an assay using surface plasmonresonance, a reduction in affinity of at least about 15%, preferably atleast about 20%, more preferably at least about 25%, more preferably atleast about 40%, more preferably at least about 50% indicatesresponsiveness: and that continuation treatment is indicated. For acompetitive Farr assay, the same reductions in affinity generally apply.For other assays, the change can be at least about any of the abovepercentages, and further can be at least about any of the followingpercentages: 75%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%.

The invention also provides methods of identifying antibody affinity (asreflected by K_(D)′ and/or its equivalent) which may be a suitable“cut-off” value for selection of an individual to receive a treatment(s)described herein. As is evident from the experimental data providedherein, a population of individuals may be assessed for initial affinitycompared to affinity after initiation of treatment (given a sufficientamount of conjugate and sufficient time to produce a response, if any,as indicated by reduction (if any) in affinity). Initial affinityvalue(s) which indicate a response (in terms of reduction of affinityand/or palliation of disease) is then determined and can be used as anindicator for selection of an individual to receive treatment (asdescribed herein).

Accordingly, the invention provides a method of identifying an antibodyaffinity which may indicate applicability (or use) of a treatment for anantibody-mediated pathology, said method comprising (a) measuringaffinity of antibodies implicated in the antibody-mediated pathologyfrom a plurality of individuals (preferably measuring the affinity foreach individual), said individuals having an antibody-mediatedpathology, said affinity being between said antibodies and an analog ofan immunogen implicated in the antibody-mediated pathology; (b)administering to the plurality of individuals the molecule comprisingthe analog; (c) correlating the extent of the antibody-mediatedpathology with the initial affinity. Generally, a positive correlationbetween an affinity and a reduction of symptoms (or some other suitableclinical endpoint) indicates that the antibody affinity is predictive ofsuitability of that treatment. Conversely, a negative correlationindicates that individuals having that affinity (or lower,) may beunsuitable for the treatment. For these methods, the molecule comprisingan analog may be any platform described herein. In some embodiments, theanalog lacks cell epitopes capable of activating T cells in anindividual. In some embodiments, the correlation is between the extentof change in antibody affinity with extent of disease, and, accordingly,clinically significant initial antibody affinity and/or change inantibody affinity is identified.

Measuring Antibody Affinity

Antibody affinity may be measured using methods known in the art whichassess degree of binding of DNA epitope to antibody. Generally, thesemethods comprise competition assays and non-competition assays. Withrespect to polynucleotide epitopes (which will be used in a conjugate tobe administered), affinity may be measured using polynucleotide alone orpolynucleotide-containing conjugates (as long as the polynucleotide andconjugate give equivalent, or at least convertible, values).

In a competition assay, varying concentrations of antibody or epitopeare reacted with epitope or antibody, and results may be expressed interms of amount of antibody (generally in terms of concentration)required to reach half-maximal binding, generally designated as IC₅₀.

Another convenient way to express affinity is apparent equilibriumdissociation constant, or K_(D)′, which reflects the titer-weightedaverage affinity of the antibody for the antibody-binding epitope on theconjugate. Antibody is generally obtained from whole blood and measured,by plasma, serum, or as an IgG fraction, and the affinity of thisfraction for the conjugate is measured. Methods of obtaining IgGfractions are known in the art and are described herein. One preferredway to measure affinity is to measure K_(D)′ based on a surface plasmonresonance assay as described in the Examples.

Another way to measure affinity is by kinetic (i.e., non-equilibrium)analysis, methods of which are known in the art. Preferably, rate ofdissociation (i.e., off rate) of antibody from epitope is measured.

Treatment Modalities

For purposes of this invention, the treatment methods used entail aconjugate comprising an non-immunogenic valency platform molecule and atleast two (i.e., two or more) dsDNA epitopes, preferably polynucleotideswhich bind to anti-dsDNA antibody from the individual. Preferably, thepolynucleotide is double stranded DNA, preferably the sequence5′-GTGTGTGTGTGTGTGTGTGT-3′. In some embodiments, the polynucleotidecomprises this sequence ((GT)₁₀), or consists essentially of thissequence.

For the methods of treatment, a conjugate is administered in an amountsufficient to effect a decrease in antibody affinity, preferably in anamount sufficient to effect reduction of one or more symptoms associatedwith lupus (especially lupus nephritis). For LJP 394, the conjugate isgenerally administered at least about 50 mg, preferably at least about100 mg, once a week, although, as discussed above, dosage could varydepending on affinity of antibody from an individual.

dsDNA Epitope

Double-stranded DNA (dsDNA) epitopes for use in the conjugates of thepresent invention may be any chemical moiety which specifically binds toa dsDNA antibody. In particular, epitopes of interest include those thatbind the anti-polynucleotide (particularly anti-double stranded DNA)antibodies that occur in systemic lupus erythematosis. Generally, butnot necessarily, the dsDNA epitopes used are polynucleotides, preferablyDNA (including DNA analogs).

Examples of suitable epitopes include, but are not limited to, thosethat bind to lupus anti-DNA antibodies (see U.S. Pat. Nos. 5,162,515;5,391,785; 5,276,013; 5,786,512; 5,726,329; 5,552,391; 5,268,454;5,633,395; 5,606,047).

The suitability of particular epitopes for binding antibodies accordingto this invention can be identified and/or confirmed using techniquesknown in the art and described herein. For example, to select theoptimum epitope from a library of small drug molecules believed to mimicthe dsDNA epitope for SLE, a family of platforms can be constructed inwhich each of the candidates is alternatively displayed on a similarplatform molecule. The composition is then tested for efficacy. Forexample, for in vivo use, an animal model is used in which there arecirculating antibodies of the undesired type, such as, for example, theBXSB mouse model system. The animals can be immunized with anappropriate epitope to initiate the antibody response, if necessary.Test candidates assembled onto a platform are then used to treatseparate animals, either by administration, or by ex vivo use, accordingto the intended purpose. The animals are bled before and aftertreatment, and the antibody levels in plasma are determined by standardimmunoassay as appropriate for the specific antibody. Efficacy of thecandidates is then assessed according to antibody affinity assaysdesigned to indicate antibodies specific for the epitope being tested.Appropriate affinity assays are described herein.

Polynucleotides may be screened for binding activity with antiseracontaining the antibodies of interest, for example, SLE antisera, by theassays described in the examples and known in the art. Examples of suchassays include competitive affinity assays, for example, a competitiveFarr assay and/or a competitive ELISA assay, and/or non-competitive,equilibrium affinity assay, such as the surface plasmon resonance (forexample, using BIACORE®) based assay described herein.

A competitive Farr assay in which binding activity may be expressed asIC₅₀ (the polynucleotide concentration in molar nucleotides resulting inhalf-maximal inhibition) is an exemplary assay. Polynucleotide duplexeshaving an IC₅₀ of less than about 500 nM, preferably less than 50 nM,are deemed to have significant binding activity and are, therefore,useful for making the conjugates of this invention.

Another appropriate assay is the non-competitive, equilibrium affinityassay described herein, in which a titer-weighted affinity isdetermined.

It is understood that, for purposes of this invention, more than onetype of dsDNA epitope(s) may be used in preparing a conjugate.Alternatively, one type (i.e., one chemical species) of an dsDNA epitopemay be used. If a polynucleotide (such as ds DNA) is used, generally thelength is greater than about 10 base pairs (bp), more preferably greaterthan about 15 bp, more preferably greater than or equal to about 20 bp.Generally, but not necessarily, the length is less than about 1 kb,preferably less than about 500 bp, preferably less than about 100 bp.

Valency Platform Molecules

Any of a variety of non-immunogenic valency platform molecules (alsocalled “platforms”) may be used in the conjugates of the invention. Manyhave been described in the art, such as polymers, and need not bedescribed herein. Preferably, the conjugates comprise a chemicallydefined valency platform molecule in which a precise valency (as opposedto an average) is provided. Accordingly, a defined valency platform is aplatform with defined structure, thus a defined number of attachmentpoints and a defined valency. Certain classes of chemically definedvalency platforms, methods for their preparation, conjugates comprisingthem and methods for the preparation of such conjugates suitable for usewithin the present invention include, but are not limited to, thosedescribed in the U.S. Pat. Nos. 5,162,515; 5,391,785; 5,276,013;5,786,512; 5,726,329; 5,268,454; 5,552,391; 5,606,047; and 5,663,395 andin commonly-owned U.S. Ser. Nos. 60/111,641 (U.S. Ser. No. 09/457,607and PCT App. No. PCT/US99/29339) and 60/138,260 (U.S. Ser. No.09/590,592 and PCT App. No. PCT/US00/15968), all of which are herebyincorporated by reference.

A platform may be proteinaceous or non-proteinaceous (i.e., organic).Examples of proteinaceous platforms include, but are not limited to,albumin, gammaglobulin, immunoglobulin (IgG) and ovalbumin. Borel et al.(1990) Immunol. Methods 126:159-168; Dumas et al. (1995) Arch. Dematol.Res. 287:123-128; Borel et al. (1995) Int. Arch. Allergy Immunol.107:264-267; Borel et al. (1996) Ann. N.Y. Acad. Sci. 778:80-87.

The valency of a chemically-defined valency platform molecule within thepresent invention can be predetermined by the number of branching groupsadded to the platform molecule. Suitable branching groups are typicallyderived from diamino acids, triamines, and amino diacids.

Preferred valency platform molecules are biologically stabilized, i.e.,they exhibit an in vivo excretion half-life often of hours to days tomonths to confer therapeutic efficacy, and are preferably composed of asynthetic single chain of defined composition. They generally have amolecular weight in the range of about 200 to about 200,000, preferablyabout 200 to about 50,000 (or less, such as 30,000). Examples of valencyplatform molecules within the present invention are polymers (or arecomprised of polymers) such as polyethylene glycol (PEG), poly-D-lysine,polyvinyl alcohol, polyvinylpyrrollidone, D-glutamic acid and D-lysine(in a ratio of 3:2). Preferred polymers are based on polyethyleneglycols (PEGs) having a molecular weight of about 200 to about 8,000.Other suitable platform molecules for use in the conjugates of theinvention are albumin and IgG.

Preferred valency platform molecules suitable for use within the presentinvention are the chemically-defined, non-polymeric valency platformmolecules disclosed in co-owned U.S. Pat. No. 5,552,391, herebyincorporated by reference. Particularly preferred homogeneouschemically-defined valency platform molecules suitable for use withinthe present invention are derivatized 2,2′-ethylenedioxydiethylamine(EDDA) and triethylene glycol (TEG). The AHAB-TEG platform used for LJP394 is described below.

Preferred platforms for dsDNA epitopes are tetrabromoacetyl compounds,and other tetravalent and octavalent valency platform molecules, such asthose described in Jones et al. (1995) J. Med Chem. 38:2138-2144; andU.S. Patent references provided above.

Additional suitable valency platform molecules include, but are notlimited to, tetraaminobenzene, heptaaminobetacyclodextrin,tetraaminopentaerythritol, 1,4,8,11-tetraazacyclotetradecane (Cyclam)and 1,4,7,10-tetraazacyclododecane (Cyclen).

In general, these platforms are made by standard chemical synthesistechniques. PEG must be derivatized and made multivalent, which isaccomplished using standard techniques. Some substances suitable forconjugate synthesis, such as PEG, albumin, and IgG are availablecommercially.

For purposes of this invention, the valency platform molecules have aminimum valency of at least two, preferably at least four, preferably atleast six, more preferably at least eight, preferably at least 10,preferably at least 12. As an upper limit, valency is generally lessthan 128, preferably less than 64, preferably less than 35, preferablyless than 30, preferably less than 25, preferably less than 24,preferably less than 20, although the upper limit may exceed 128.Conjugates may also have valency of ranges of any of the lower limits of2, 4, 6, 8, 10, 12, 16, with any of the upper limits of 128, 64, 35, 30,25, 24, 20.

In some embodiments, the valency platform molecule comprises a carbamatelinkage, i.e., —O—C(═O)—N<). Such platforms are described in a co-ownedpatent application entitled “Valency Platform Molecules ComprisingCarbamate Linkages” U.S. Ser. No. 60/111,641 (U.S. Ser. No. 09/457,607and PCT App. No. PCT/US99/29339), hereby incorporated by reference.

In other embodiments, valency platforms may be used which, whenconjugated, provide an average valency (i.e., these platforms are notprecisely chemically defined in terms of their valency). Examples ofsuch platforms are polymers such as linear PEG; branched PEG; star PEG;polyamino acids; polylysine; proteins; amino-functionalized solublepolymers.

Conjugation of dsDNA Epitope(s) With Valency Platform Molecules

Conjugation of a biological or synthetic molecule to thechemically-defined platform molecule may be effected in any number ofways, typically involving one or more crosslinking; agents andfunctional groups on the biological or synthetic molecule and valencyplatform molecule. Examples of standard chemistry which may be used forconjugation include, but are not limited to: 1) thiol substitution; 2)thiol Michael addition; 3) amino alkyation; 4) disulfide bond formation.

The synthetic polynucleotide duplexes that are coupled to the valencyplatform molecule are composed of at least about 20 bp and preferably20-50 bp. Polynucleotides described herein are deoxyribonucleotidesunless otherwise indicated and are set forth in 5′ to 3′ orientation.Preferably the duplexes are substantially homogeneous in length; thatis, the variation in length in the population will not normally exceedabout ±90%, preferably ±10%, of the average duplex length in base pairs.They are also preferably substantially homogeneous in nucleotidecomposition; that is, their base composition and sequence will not varyfrom duplex to duplex more than about 10%. Most preferably they areentirely homogeneous in nucleotide composition from duplex to duplex.

Based on circular dichroic (CD) spectra interpretation, duplexes thatare useful in the invention assume a B-DNA type helical structure. Itshould be understood that it is not intended that the invention belimited by this belief and that the duplexes may, upon more conclusiveanalysis assume Z-DNA and/or A-DNA type helical structures.

These polynucleotide duplexes may be synthesized from native DNA orsynthesized by chemical or recombinant techniques. Naturally occurringor recombinantly produced dsDNA of longer length may be digested (e.g.,enzymatically, chemically and/or by mechanical shearing),andfractionated (e.g., by agarose gel or Sephadex™ column) to obtainpolynucleotides of the desired length.

Alternatively, pairs of complementary single-stranded polynucleotidechains up to about 70 bases in length are readily prepared usingcommercially available DNA synthesizers and then annealed to formduplexes by conventional procedures. Synthetic dsDNA of longer lengthmay be obtained by enzymatic extension (5′-phosphorylation followed byligation) of the chemically produced shorter chains.

The polynucleotides may also be made by molecular cloning. For instance,polynucleotides of desired length and sequence are synthesized as above.These polynucleotides may be designed to have appropriate termini forligation into specific restriction sites. Multiple iterations of theseoligomers may be ligated in tandem to provide for multicopy replication.The resulting construct is inserted into a standard cloning vector andthe vector is introduced into a suitable microorganism/cell bytransformation. Transformants are identified by standard markers and aregrown under conditions that favor DNA replication. The polynucleotidesmay be isolated from the other DNA of the cell/microorganism bytreatment with restriction enzymes and conventional sizefractionation.(e.g., agarose gel, Sephadex™ column).

Alternatively, the polynucleotides may be replicated by the polymerasechain reaction (PCR) technology. Saiki et al (1985) Science230:1350-1354;. Saiki et al. (1988) Science 239:487-491; Sambrook et al.(1989) p 14.1-14.35.

The polynucleotides are conjugated to the chemically-defined valencyplatform molecule in a manner that preserves their antibody bindingactivity. This is done, for example, by conjugating the polynucleotideto the valency platform molecule at a predetermined site on thepolynucleotide chain such that the polynucleotide forms a pendant chainof at least about 20 base pairs measured from the conjugating site tothe free (unattached) end of the chain.

In one embodiment, the polynucleotide duplexes are substantiallyhomogenous in length and one strand of the duplex is conjugated to thevalency platform molecule either directly or via a linker molecule.Synthetic polynucleotides may be coupled to a linker molecule beforebeing conjugated to a valency platform molecule. Usually the linkercontaining strand of the duplex is coupled at or proximate (i.e., withinabout 5 base pairs) to one of its ends such that each strand forms apendant chain of at least about 20 base pairs measured from the site ofattachment of the strand to the linker molecule. The second strand isthen annealed to the first strand to form a duplex. Thus, a conjugatewithin the present invention may be generally described by the followingformula:[(PN)_(n)-linker]_(m)-valency platform moleculewherein PN=a double-stranded polynucleotide with “n” nucleotides,wherein n=at least about 20 and m=2-8.

In one embodiment, the polynucleotides of the conjugates are coupled toa linker molecule at or proximate one of their ends. The linker moleculeis then coupled to the chemically-defined valency platform molecule. Asdescribed in U.S. Pat. No. 5,552,391 and incorporated herein byreference, exemplary of suitable linker molecules within the presentinvention are 6 carbon thiols such as HAD, a thio-6 carbon chainphosphate, and HAD_(p)S, a thio-6 carbon chain phosphorothioate.Chemically-defined valency platform molecules within the presentinvention are formed, for example, by reacting amino modified-PEG with3,5-bis-(iodoacetamido)benzoyl chloride (hereinafter “IA-DABA”);3-carboxypropionamide-N,N-bis-[(6′-N′-carbobenzyloxyaminohexyl)acetamide]4″-nitrophenyl ester (hereinafter “BAHA”);3-carboxypropionamide-N,N-bis-[(8′-N′-carbobenzyloxyamino-3′,6′-dioxaoctyl)acetamide]4″-nitrophenyl ester (hereinafter “BAHA_(ox)”); or by reactingPEG-bis-chloroformate withN,N-di(2-[6′-N′-carbobenzyloxyaminohexanoamido]ethyl)amine (hereinafter“AHAB”) to form chemically-defined valency platform molecules.

For example, a defined double-stranded polynucleotide (PN) can beconjugated to a valency platform molecule by first providing a singlechain consisting of approximately 20 alternating cytosine (C) andadenosine (A) nucleotides. Four CA chains may then be covalentlyconjugated through linkers such as HAD to four reactive sites on aderivatized platform molecule such as triethylene glycol. The valencyplatform molecule is synthesized to include groups such as bromoacetyl.During the conjugation, a leaving group is displaced by sulfur. A secondsingle nucleotide chain consisting of approximately 20 alternatingthymidine (T) and guanosine (G) nucleotides can then be annealed to theCA strand to form a double-stranded PN conjugate of the formula,[(PN)₂₀-linker]₄ -valency platform molecule.

Alternatively, in another embodiment, the polynucleotide may be coupledto the derivatized valency platform molecule at the 3′ end of thepolynucleotide via a morpholino bridge formed by condensing an oxidized3′ terminal ribose on one of the strands of the polynucleotide with afree amino group on the derivatized platform molecule and thensubjecting the adduct to reducing conditions to form the morpholinolinkage, as described in U.S. Pat. No. 5,553,391. Such coupling requiresthe derivatized platform molecule to have at least an equal number ofamino groups as the number of polynucleotide duplexes to be bound to theplatform molecule. The synthesis of such a conjugate is carried out intwo steps. The first step is coupling one strand of the polynucleotideduplex to the derivatized platform molecule via a condensation/reductionreaction. The oxidized 3′ terminal ribose is formed on the singlepolynucleotide strand by treating the strand with periodate to convertthe 3′ terminal ribose group to an oxidized ribose group. Thesingle-stranded polynucleotide is then added slowly to an aqueoussolution of the derivatized platform molecule with a pH of about 6.0 to8.0 at 2-8° C.

The molar ratio of polynucleotide to platform molecule in all theconjugation strategies will normally be in the range of about 2:1 toabout 30:1, usually about 2:1 to about 8:1 and preferably about 4:1 to6:1. In this regard, it is preferable that the conjugate not have anexcessively large molecular weight as large molecules, particularlythose with repeating units, of m.w.>200,000 may be T-independentimmunogens. See Dintzis et al. (1983) J. Immunol. 131:2196 and Dintziset al. (1989) J. Immunol. 143:1239. During or after the condensationreaction (normally a reaction time of 24 to 48 hr), a strong reducingagent, such as sodium cyanoborohydride, is added to form the morpholinogroup. The complementary strand of the duplex is then added to theconjugate and the mixture is heated and slowly cooled to cause thestrands to anneal. The conjugate may be purified by gel permeationchromatography.

An alternative to the ribose strategy is forming aldehydefunctionalities on the polynucleotides and using those functionalitiesto couple the polynucleotide to the platform molecule via reactivefunctional groups thereon. Advantage may be taken of the fact that gemvicinal diols, attached to the 3′ or 5′ end of the polynucleotide, maybe oxidized with sodium periodate to yield aldehydes which can condensewith functional amino groups of the platform molecule. When the diolsare in a ring system, e.g., a five-membered ring, the resultingcondensation product is a heterocyclic ring containing nitrogen, e.g., asix-membered morpholino or piperidino ring. The imino-condensationproduct is stabilized by reduction with a suitable reducing agent; e.g.,sodium borohydride or sodium cyanoborohydride. When the diol is acyclic,the resulting oxidation product contains just one aldehyde and thecondensation product is a secondary amine.

Another procedure involves introducing alkylamino or alkylsulfhydrylmoieties into either the 3′ or 5′ ends of the polynucleotide byappropriate nucleotide chemistry, e.g., phosphoramidite chemistry. Thenucleophilic groups may then be used to react with a large excess ofhomobifunctional cross-linking reagent, e.g., dimethyl suberimidate, inthe case of alkylamine derivatives, or an excess of heterobifunctionalcross-linking reagent, e.g., m-maleimidobenzoyl-N-hydroxysuccinimideester (MBS) or succinimidyl (4-iodoacetyl) aminobenzoate (SIAB), for thealkylsulfhydryl derivatives. Once excess cross-linker is removed, thepolynucleotide derivatives are reacted with amino groups on the platformmolecule. Alternatively, the sulfhydryl group may be reacted with anelectrophilic center on the platform, such as a maleimide orα-haloacetyl group or other appropriate Michael acceptor.

Still another strategy employs modified nucleosides. Suitabledeoxynucleoside derivatives can be incorporated, by standard DNAsynthetic chemistry, at desired positions in the polynucleotide,preferably on the 5′ or 3′ ends. These nucleoside derivatives may thenreact specifically and directly with alkylamino groups on the platformmolecule. Alternatively, side reactions seen with the above-describeddialdehyde chemistry, such as amine catalyzed beta-elimination, can becircumvented by employing appropriate nucleoside derivatives as the 3′terminus of the chain to be attached. An example of this is 5′ methyleneextension of ribose; i.e., a 5′ (2-hydroxyethyl)-group instead of a 5′hydroxymethyl group. An alternative would be to use a phosphonate orphosphinate linkage for the 3′ terminal dinucleotide of thepolynucleotide to be attached to the platform molecule.

A description of the synthesis of the conjugate LJP 394, a tetravalentconjugate, is described in Jones et al. (1995) and in U.S. Pat. No.5,552,391, which are hereby incorporated by reference. LJP 394 comprisesfour 20-mer oligonucleotides consisting of alternating C and Anucleotides, (CA)₁₀, attached to a platform and annealed withcomplementary 20-mer oligonucleotides consisting of alternating G and Tnucleotides, (GT)₁₀, oligonucleotide. The valency platform molecule usedin LJP 394 is shown immediately below.

Administration of Conjugates

Various formulations of epitope-presenting conjugate(s) may be used foradministration. In some embodiments, the epitope-presenting conjugate(s)may be administered neat. In some embodiments, the compositions comprisea conjugate(s) and a pharmaceutically acceptable excipient, and may bein various formulations. Pharmaceutically acceptable excipients areknown in the art, and are relatively inert substances that facilitateadministration of a pharmacologically effective substance. For example,an excipient can give form or consistency, or act as a diluent. Suitableexcipients include but are not limited to stabilizing agents, wettingand emulsifying agents, salts for varying osmolarity, encapsulatingagents, buffers, and skin penetration enhancers. Excipients as well asformulations for parenteral and nonparenteral drug delivery are setforth in Remington's Pharmaceutical Sciences 19th Ed. Mack Publishing(1995).

Generally, these compositions are formulated for administration byinjection (e.g., intraperitoneally, intravenously, subcutaneously,intramuscularly, etc.). Accordingly, these compositions are preferablycombined with pharmaceutically acceptable vehicles such as saline,Ringer's solution, dextrose solution, and the like. Generally, theconjugate will normally constitute about 0.01% to 10% by weight of theformulation due to practical, empirical considerations such assolubility and osmolarity. The particular dosage regimen, i.e., dose,timing and repetition, will depend on the particular individual and thatindividual's medical history. Generally, a dose of about 1 μg to about100 mg conjugate/kg body weight, preferably about 100 μg to about 10mg/kg body weight, preferably about 150 μg to about 5 mg/kg body weight,preferably about 250 μg to about 1 mg conjugate/kg body weight.Empirical considerations, such as the half life, generally willcontribute to determination of the dosage. Other dosages, such as about50 to 100 mg per week, are also described herein. If used as atoleragen, conjugate may be administered daily, for example, in order toeffect antibody clearance (pheresis), followed by less frequentadministrations, such as two times per week, once a week, or even lessfrequently. Frequency of administration may be determined and adjustedover the course of therapy, and is based on maintaining tolerance (i.e.,reduced or lack of immune response to dsDNA). Other appropriate dosingschedules may be as frequent as continuous infusion to daily or 3 dosesper week, or one dose per week, or one dose every two to four weeks, orone dose on a monthly or less frequent schedule depending on theindividual or the disease state. Repetitive administrations, normallytimed according to B cell turnover rates, may be required to achieveand/or maintain a state of humoral anergy. Such repetitiveadministrations generally involve treatments of about 1 μg to about 10mg/kg body weight or higher every 30 to 60 days, or sooner, if anincrease in anti-dsDNA antibody level is detected. Alternatively,sustained continuous release formulations of the compositions may beappropriate. Various formulations and devices for achieving sustainedrelease are known in the art.

Other formulations include those suitable for oral administration, whichmay be suitable if the conjugate is able to cross the mucosa. Similarly,an aerosol formulation may be suitable.

Other formulations include suitable delivery forms known in the artincluding, but not limited to, carriers such as liposomes. Mahato et al.(1997) Pharm. Res. 14:853-859. Liposomal preparations include, but arenot limited to, cytofectins, multilamellar vesicles and unilamellarvesicles.

In some embodiments, more than one conjugate may be present in acomposition. Such compositions may contain at least one, at least two,at least three, at least four, at least five different conjugates. Such“cocktails”, as they are often denoted in the art, may be particularlyuseful in treating a broader range of population of individuals. Theymay also be useful in being more effective than using only one (or fewerthan are contained in the cocktail) conjugate(s).

The compositions may be administered alone or in conjunction with otherforms of agents that serve to enhance and/or complement theeffectiveness of a conjugate of the invention, including, but notlimited to, anti-helper T cell treatments. Such treatments usuallyemploy agents that suppress T cells such as steroids or cyclosporin;Another agents are corticosteroid and/or cyclophosphamideimmunosuppressive therapy.

Detection and measurement of indicators of efficacy are generally basedon measurement of anti-double-stranded DNA antibody and/or clinicalsymptoms associated with SLE, which are known in the art.

Lupus nephritis (kidney glomerulonephritis or kidney inflammation) ischaracterized by a progressive loss of kidney function culminating inrenal failure. Lupus nephritis is characterized by hematuria, decreasedurine output, elevated blood urea nitrogen levels, elevated serumcreatinine levels, hypertension, and proteinuria. Accordingly, theseparameters can be monitored as a means of monitoring kidneydegeneration.

Compositions of the Invention

Kits Comprising Epitopes Which Bind to Anti-ds DNA Antibodies

The invention also provides kits for measuring antibody affinities foruse in the methods described herein, particularly affinity for anepitope which binds to anti-ds DNA antibodies. Accordingly, theinvention includes kits containing (i.e., comprising) one or more dsDNAepitopes, preferably polynucleotides (preferably, double stranded (ds)DNA molecules) comprising an epitope which binds to an anti-ds DNAantibody from an individual (and the epitope-containing polynucleotidebinds to an anti-ds DNA antibody from an individual). Accordingly, thekits comprise a molecule or moiety comprising a ds DNA epitope, such asany described herein. In one embodiment, the kit comprises apolynucleotide with (comprising) the sequence (or, alternatively,consisting essentially of or consisting of the sequence)5′-GTGTGTGTGTGTGTGTGTGT-3′. Kits comprising a polynucleotide(s) or anyother suitable ds DNA epitope may further include instructions for usingthe polynucleotide to detect affinity of an individual's anti-ds DNAantibody(ies) for the polynucleotide (or ds DNA epitope). In otherembodiments, the kits comprise the conjugates described herein, withinstructions for using the conjugate to detect affinity of anindividual's anti-ds DNA antibodies for the conjugate. Preferably, theconjugate is LJP 394.

The kits may be used, for example, to test an individual to determine ifthe individual is suitable or unsuitable for treatment with theconjugate(s), as well as for monitoring purposes. The kits may also beused in determining affinity cut-off values (i.e., affinity values whichcorrelate with clinical results). The kits of this invention are insuitable packaging, and may optionally provide additional componentssuch as, buffers and instructions for determining affinity or binding toanti-dsDNA antibody, such as capture reagents, developing reagents,labels, reacting surfaces, means for detection, control samples, andinterpretive information. The instructions may be for any measurement ofantibody affinity, including, but not limited to, those assays describedherein. Accordingly, in some embodiments, the instructions are fordetermining affinity using surface plasmon resonance. In otherembodiments, the instruction are for determining affinity using directbinding assays and/or Farr assays. In some embodiments, reagentsdescribed above are supplied such that multiple measurements may bemade, such as allowing for measurements in the same individual over timeor multiple individuals.

Generally, the dsDNA epitope(s) of the kit, preferably apolynucleotide(s) of the kit (whether in free form or attached to aconjugate or other matrix), contains, or alternatively consists of, theepitope that will be or is used in treatment, or has been demonstratedto have about the same affinity for an individual's anti-ds DNAantibodies as the epitope(s) that will be used in treatment. In otherembodiments, the kits comprising a ds DNA epitope whose affinity foranti-dsDNA antibodies mimics or alternatively can be correlated to thatof the dsDNA epitope to be used in treatment, such as5′-GTGTGTGTGTGTGTGTGTGT-3′. These dsDNA epitopes can be used as“proxies” for the ds DNA epitope to be used in treatment, such as LJP394, in assessing antibody affinity for the methods described herein.

Any appropriate means for detecting binding of the antibodies may beemployed (and provided in the kits) such as a labeled anti-humanantibody, when the presence of human anti-dsDNA antibodies is tested,wherein the label may be an enzyme, fluorophore, chemiluminescentmaterial radioisotope or coenzyme. Generally, the label used will be anenzyme. Accordingly, in some embodiments, the kit(s) of the inventionfurther comprises a label. In some embodiments, the polynucleotide inthe kit(s) is conjugated to biotin. In a preferred embodiment, the dsDNAepitope (such as a polynucleotide, for example, double stranded DNA) isbiotinylated. Biotinylation may also be accomplished using commerciallyavailable reagents (i.e., Pharmacia; Uppsala, Sweden). In anotherpreferred embodiment, the biotinylated dsDNA epitope comprises, consistsessentially or, or consists of is 5′-GTGTGTGTGTGTGTGTGTGT-3′.

In other embodiments, the invention provides a kit comprising (a) aconjugate as described herein, such as LJP 394; and (b) a polynucleotide(or other ds DNA epitope) used in the conjugate, or, alternatively, apolynucleotide comprising the polynucleotide used in the conjugate (or amolecule or moiety comprising the epitope to be used in the conjugate).When used for affinity measurements, the conjugate and/or polynucleotidemay be biotinylated. In some embodiments, the kit contains instructionsfor administering the conjugate to an individual as well as instructionsfor using the conjugate and/or the polynucleotide (including apolynucleotide comprising the polynucleotide used in the conjugate) fordetecting affinity for an antibody in an individual which binds to dsDNA as described herein. As discussed herein, a combination of aconjugate to be used for treatment and a molecule comprising a ds DNAepitope, the binding activity or affinity of which mimics, or can becorrelated with, the epitope of the conjugates is used in the kits.

In other embodiments, the invention provides a kit comprising an analogwhich binds to an antibody implicated in an antibody mediated pathology.In some embodiments, the invention provides a kit comprising a moleculecomprising an epitope which binds to an antibody implicated in anantibody mediated pathology. These kits, also useful for making affinitydeterminations, are in suitable packaging and optionally includeinstructions for using the analog or molecule in the kit to determineaffinity of an antibody implicated in an antibody-mediated pathologywith the analog or epitope-containing molecule. Peptide analogs whichbind to antibodies implicated in antiphospholipid syndome (APS) andlupus are disclosed in WO 96/40197 and WO 97/46251. Polypeptides whichbind to antibodies implicated in antibody-mediated pathology havingspecificity for domain 1 of β₂GPI are disclosed in commonly owned U.S.Ser. No. 09/328,199 (PCT/US99/13194). Other analogs andepitope-containing molecules are known in the art. In anotherembodiment, the kit comprises D-galactopyranoside, or, alternatively, amolecule which exhibits specific binding to an anti-αGal (galactose α1,3galactosyl) antibody and instructions and/or-reagents for determiningantibody affinity.

The following Examples are provided to illustrate but not limit theinvention.

EXAMPLES Example 1 Inhibition of Binding of Anti-dsDNA Antibodies to DNAby LJP 394

After determining the presence of anti-ds DNA antibodies in patientsusing a Farr assay, a competitive Farr assay was used to measure theaffinity of anti-dsDNA antibodies found in sera from patients with SLEto LJP 394. In addition, the assay was used to measure the affinity ofanti-dsDNA antibodies found in sera from three animals models of SLE(BXSB mice, NZB×NZW F₁ mice, and MRL/lpr mice).

The Farr assay used ¹²⁵I-labeled recombinant dsDNA (Diagnostic ProductsCorporation, Los Angeles, Calif.) that was combined with the anti-dsDNAantibodies found in sera from patients with SLE or from the mouse modelsof SLE. Anti-dsDNA antibodies were obtained from serum samples of donorswith SLE collected through a volunteer donor program. Blood samples weredrawn, serum harvested, aliquots made, labeled, and stored frozen at−70° C. until used. In this assay, 25 μl of patient's serum was added to75 μl of Tris buffer (50 mM Tris, 150 mM NaCl pH 7.5, 10% normal rabbitserum), then 100 μl of ¹²⁵I-labeled recombinant dsDNA was added, mixedand incubated at 37° C. for one hour. Similar samples containing knownamounts of anti-dsDNA antibodies (calibrators) were prepared andincubated at the same time. 500 μl of 70% saturated ammonium sulfate wasadded to each tube, mixed, and then centrifuged at 800×g for 15 minutesto precipitate the antibodies in solution. The supernatant was decantedand the amount of radioactivity in the precipitated product wasdetermined by counting the radioactivity in a gamma counter. The amountof radioactivity in the precipitant is proportional to the amount ofanti-dsDNA antibodies that bound to ¹²⁵I-labeled recombinant dsDNA.Calibrators with known amounts of anti-dsDNA antibodies were used togenerate a standard curve from which the amount of dsDNA binding byanti-dsDNA antibodies could be calculated.

Serum samples from 58 patients were assayed-for the presence ofantibodies to dsDNA using the Farr assay described above. Forty-two ofthese samples had sufficient levels of antibody (≧20% binding) to use inthe LJP 394 inhibition assay.

LJP 394 was tested for its ability to inhibit binding of anti-dsDNAantibodies to ¹²⁵I-labeled recombinant dsDNA by a competitive Farrassay. Calf thymus DNA (ctDNA) was also used in the inhibition assay asanother source of dsDNA. Calf thymus dsDNA was prepared by dissolvingcalf thymus DNA in nuclease-S1 buffer (0.2 M NaCl, 50 mM sodium acetatepH 4.5, 1 mM ZnSO₄ and 0.5% glycerol) and 100,000 units of S-1 nucleaseand incubating for one hour at 37° C. The dsDNA was extracted from thismixture by adding an equal volume of phenol-chloroform, mixing,centrifuging, and harvesting the aqueous layer. The dsDNA was thenprecipitated by adding 2 volumes of EtOH, mixing, and centrifuging. Thepellet was harvested, dried under vacuum and dissolved in water toapproximately 10 mg/ml. The final concentration of the ct DNApreparation was determined spectrophotometrically assuming an extinctioncoefficient of 33 μg per 1 OD unit at 260 nM.

Each serum sample that gave ≧20% binding was tested in the inhibitionassay. Briefly, 25 μl of patient's serum was added to 75 μl of Trisbuffer (50 mM Tris, 150 mM NaCl pH 7.0, 10% normal rabbit serum)containing various concentrations of inhibitor (either calf thymus dsDNAor LJP 394), then 100 μl of ¹²⁵I-labeled recombinant dsDNA was added,mixed and incubated at 37° C. for one hour. 500 μl of 70% saturatedammonium sulfate was added to each tube, mixed and then centrifuged at800×g for 15 minutes. The supernatant was decanted and the amount ofradioactivity in the precipitated product was determined by counting theradioactivity in a gamma counter. Extent of inhibition was calculated bythe following formula: {[(cpm patient's serum without inhibitor−cpmwithout patient's serum, no inhibitor)−(cpm patient's serum withinhibitor−cpm without patient's serum, no inhibitor)] divided by (cpmpatient's serum without inhibitor−cpm without patient's serum, noinhibitor)} all times 100.

FIGS. 1A-C illustrate the ability of LJP 394 to inhibit the binding ofautoantibodies from a representative populations of patients with SLE.Overall, LJP 394 was capable of inhibiting binding of the autoantibodiesto dsDNA in 42 out of 42 patients with SLE. The inhibition curves forLJP 394 and calf thymus dsDNA were parallel, suggesting that theantigenic determinants being recognized by the SLE sera were identicalon both the calf thymus dsDNA and LJP 394.

The ability of LJP 394 to inhibit the binding of anti-DNA antibodies ina mouse models of SLE was also tested. Competitive inhibition assayswith calf thymus dsDNA and LJP 394 were performed as described above andthe results are shown in Table 1. The 50% inhibition ratios (IC₅₀ LJP394/IC₅₀ ctDNA) were lowest for human anti-dsDNA antibodies (from SLEsera), compared to the mouse antibodies. LJP 394 showed high affinityfor human antibodies and the NZB×NZW F1 mouse strain. TABLE 1Competitive Inhibition of Binding of Anti-ds DNA Antibodies by ctDNA andLJP 394 IC₅₀ No. IC₅₀, μg/ml (mean ± SD) LJP 394/ of sera Source of seractDNA LJP 394 ctDNA ratio 3 MRL 0.356 ± 0.455 200 ± 42  562(lpr/lpr)(mouse) 3 NZBxNZWF₁ 0.021 ± 0.011 5.5 ± 0.7 258 (mouse) 5 BXSB(mouse) 0.028 ± 0.000 215 ± 144 7679 42 Human SLE  1.88 ± 0.920 46 ± 1624

Example 2 Identifying SLE Patients by Affinity Assay

SLE patients were readily identified by measuring the binding affinityof sera from SLE patients to LJP 394 dsDNA epitope using the surfaceplasmon resonance assay described below. IgG fractions from 10 SLEpatients serum samples and 10 normal patient serum samples wereevaluated for binding to LJP 394 ds DNA epitope with a non-competitivedirect affinity assay (BIACORE™; Piscataway, N.J.), as disclosed herein.FIG. 2 illustrates that the SLE samples all showed saturable binding tothe epitope while normal samples showed low binding to the LJP 394epitope and low specific binding.

Example 3 Determination of Titer-Weighted Average Affinity of Antibodiesfor Conjugate and Response to Treatment with Conjugate

An assay using surface plasmon resonance was developed to directlymeasure a titer-weighted average affinity of antibodies from SLEpatients for the conjugate LJP 394. Surface plasmon resonance is used toquantify the fractional saturation of antigen with antibody. This assaywas adapted so that it measured the average affinity of the IgGpopulation of LJP 394.

Materials and Methods

Reagents. Streptavidin CM5 chips, HBS buffer (0.01 M HEPES pH 7.4, 0.15M NaCl, 3 mM EDTA and 0.005% (v/v) surfactant P20) were obtained fromBIACORE AB (Piscataway, N.J.).

LJP 394 is composed of four 20-mer dsDNA epitopes that are covalentlyattached to a triethyleneglycol-based platform by a thiol linkage. TheDNA epitope was composed of 5′-(CA)₁₀-3′ strands annealed tocomplementary GT strands, with biotin attached at the free 5′ ends ofthe GT strand. Biotin was incorporated by using Biodite biotin amidite(Pharmacia; Uppsala, Sweden) in the final coupling of the (GT)₁₀ strand.LJP 394 was prepared essentially as described in Jones et al. (1995)except that this biotin-modified (GT)₁₀ strand was used in the annealingstep. In some experiments, only the dsDNA epitope was used forimmobilization on the streptavidin chip. The epitope was prepared byannealing 5′-(CA)₁₀-3′ to 5′-biotin-(TG)₁₀-3′ and purifying the dsDNA byHPLC.

Plasma samples from SLE patients were collected at visit 2, prior tostart of study drug administration and at visit 11, after 4 months ofweekly drug administration.

Total IgG fraction was isolated from plasma by combining 100 μl ofplasma with 100 μl of IgG binding buffer (Pierce Chemical Co.; Rockford,Ill.) and mixing with Immunopure Plus® protein G agarose beads (PierceChemical Co.) according to manufacturer recommendations. Elution of IgGfrom the beads was accomplished by following the acidelution/neutralization protocol of Pierce Chemical Co., and 300 μl ofacid eluted IgG was neutralized with 100 μl of 1 M NaPO₄, pH 7.5. Thesepurified IgG samples were then used in the titration experiments. TotalIgG concentrations were determined with the Bradford assay (Biorad;Hercules, Calif.).

Surface Plasmon Resonance. All experiments were performed using aBIACORE® 2000 instrument at 25° C. with a flow rate of 10 μl/minute. LJP394 was attached to the streptavidin CM5 chip through its 5′ biotingroup by flowing a 50 μg/ml solution of LJP 394 in HBS+0.3 M NaCl overthe chip for 20 minutes at 5 μl/minute. The chip waspreconditioned-prior to titration with 3×1 minute pulses of regenerationbuffer (1M NaCl and 50 mM NaOH). When the dsDNA epitope of LJP 394 wasused for immobilization, the biotinylated epitope was flowed over thechip at a concentration of 10 μg/ml using similar conditions as employedfor the biotinylated LJP 394 epitope.

Antibody titrations of the dsDNA (LJP 394) chip were performed withserial 1:2 dilutions of purified IgG in HBS. Sample was injected for 5minutes, which is adequate association time for a significant approachto the response plateau, and was followed by a 4 minute dissociationperiod where HBS is flowed over the chip, then a 30 second regenerationwas performed with 1 M NaCl, 50 mM NaOH.

Analysis. Response plateau values (R_(eq)) were obtained by a nonlinearleast squares fit of the association curves to equation 1, aftersubtraction of a background curve for an empty flow cell, to account forbulk response/buffer effect, and using the manufacturers software(BiaEvaluation version 2.2, Uppsala, Sweden)R _(t) =R _(eq)(1−e ^(−ks(t−t0)))+R ₀   (equation 1)where R_(t) is the measured response at time t, R_(eq) is theequilibrium plateau response, t is time, t₀is initial time, k_(s) is anapparent association constant (k_(s)=k_(a)C+k_(dis), where k_(a) is theassociation constant, C is the analyte concentration and k_(dis) is thedissociation constant), and R₀ is a response offset. These responseplateaus were plotted versus the concentration of total IgG, and fittedto equation 2 to obtain values for R_(max) and K_(d)*. $\begin{matrix}{R_{eq} = \frac{R_{\max}A_{T}}{K_{d}^{*} + A_{T}}} & \left( {{equation}\quad 2} \right)\end{matrix}$where A_(T) is the total antibody (IgG) concentration, R_(max) is themaximum response plateau and K_(d)* is an apparent dissociationconstant. K_(d)* is the same as <K_(d)′> in equation 3 (below), thetiter-weighted-average (TWA) dissociation constant. The derivation ofK_(d)′ was performed as described in Sem et al. (1999) and providesinsight into the physical meaning of the K_(d)* constant in equation 2.This analysis pertains to the case of a polyclonal pool of n differentantibody, subpopulations, where B=LJP 394 and A_(i)=antibodysubpopulation i. $\begin{matrix}{{< K_{d}^{\prime}>=\frac{A_{T}}{\sum\limits_{i = 1}^{n}\left( {A_{i}/K_{i}} \right)}} = \frac{1}{\sum\limits_{i = 1}^{n}\left( {r_{i}/K_{i}} \right)}} & \left( {{equation}\quad 3} \right)\end{matrix}$where r_(i) (relative titer) is the fraction of total antibody presentas form i, defined as r_(i)=A_(i)/A_(T). Thus, equation 3 is the generalequation describing the observed dissociation constant for a polyclonalpopulation of n different antibody subpopulations of relative titer(fractional presence) r_(i) and dissociation constant K_(i). This<K_(d)′> is the apparent K_(d) of eq 2, K_(d)*.

The measured apparent dissociation constant K_(d)′ reflects bothinherent affinity of antibody subpopulation i for antigen, and relativetiter of antibody subpopulation i (r_(i)). In general, 0<r_(i)<1, soK_(d)′>K_(i). That is, the factors that can cause K_(d)′ to decrease arean increase in affinity (K_(i) decreases) and/or an increase in relativetiter of antibody subpopulation i (r_(i) increases). In practice, in apolyclonal population of antibodies, there will be many differentantibody subpopulations that bind, each with slightly differentaffinity.

The above analysis, and that further described in Sem et al. (1999),produces an apparent dissociation constant that is a reflection of thevarious affinities and titers of clonally related subpopulations ofantibodies within a polyclonal pool. The apparent dissociation constantobtained as described is the titer-weighted-average (TWA) dissociationconstant derived in equation 3, <K_(d)′>. The value of <K_(d)′> isdominated by antibody subpopulations that have the largest r_(i)(highest relative titer) and smallest K_(i) (highest affinity) incombination. Any change in relative titers of subpopulations with agiven affinity will change the apparent dissociation constant accordingto equation 3.

Results

Study 1

Equilibrium binding values from 4 dilutions of plasma IgG from SLEpatients were evaluated with the surface plasmon resonance-basedaffinity assay to determine the concentration of IgG required to reachhalf-maximal binding. This value, the apparent equilibrium dissociationconstant (K_(d)′ in mg/ml of IgG) reflects the titer-weighted averageaffinity of the IgG population for LJP 394, as outlined above and by Semet al. (1999).

Table 2 contains a comparison of the Kd′ from the initial IgG affinityfor LJP 394 in naive patients (visit 2) and the affinity after 4 monthsof weekly study drug administration (visit 11). Data from this firstpatient population indicated that patients who entered the trial withhigh affinity antibodies for LJP 394 (low numeric K_(d)′=high affinity)were more likely to respond to drug treatment (higher numericK_(d)′=decrease in TWA). TABLE 2 Comparison of K_(D)′ values PatientK_(d)′ @visit 2 K_(d)′ @ % change ID (mg/ml) visit 11(mg/ml) in K_(d)′02-19 0.05 0.32 540 08-76 0.15 0.34 127 04-37 0.41 0.77 87 02-13 0.510.88 73

The data in Table 2 suggested that the initial IgG affinity (K_(d)′) maypredict the response of the patients to LJP 394. When IgG affinities ofthe same samples were measured using the (non-competitive) Farr assay asdescribed in Example 1, the initial Farr values were not predictive ofpatient response, as shown in Table 3. TABLE 3 Comparison of Farr valuesPatient Farr @visit 2 Farr @ % change ID (IU/ml) visit 11 (IU/ml) inFarr 02-19 35 14 −60 08-76 60 27 −55 04-37 23 18 −22 02-13 75 72 −4

FIG. 3A depicts the dose dependent decrease in anti-dsDNA antibodyburden (K_(d)′) in SLE patients from treatment with LJP 394. FIG. 3Bdepicts the same data in the form of a bar graph. These resultsdemonstrate the dose dependent effect of LJP 394 at 50 mg/wk provided abetter response than 10 mg/wk and both groups were different thanplacebo.

Study 2

For the second SLE patient population studied, the first plasma samplewas obtained at visit 2 or 3, prior to start of study drugadministration, and the second sample was collected immediatelyfollowing 4 months of study drug administration at 100 mg/wk (visit 18or 19). These 2 samples were then used to determine the initial affinityin naive patients and the affinity after 4 months of study drugadministration. There was no known bias to the selection of samplesexcept that they included all the patients available for those sampletimes. The results are shown in Table 4 (number of available samplesindicates number of samples available for analysis; total number offlares represents only those flares attributable to lupus). Table 5includes all the samples in Table 4 in addition to more patient samples.TABLE 4 Samples for Study 2 # available samples/ # of flaresrepresented/ total # patients total # of flares LJP 394 71/114 16/19Placebo 70/115 16/23

TABLE 5 Samples for Study 2 # available samples/ # of flaresrepresented/ total # patients total # of flares LJP 394 104/114 18/19Placebo 106/115 23/23

Change in antibody affinity. Results for the drug treatment group andfrom the placebo group are shown in FIGS. 4 and 5. Each squarerepresents data from a single patient. FIGS. 4A and 4B reflect thepatient pool summarized in Table 4. FIGS. 4C and 4D reflect the patientpool summarized in Table 5. FIGS. 5A and 5B display the data shown inFIGS. 4A and 4B in terms of percent change in Kd′. V3pre K_(d)′represents the K_(d)′ for patients just prior to the start of study drugadministration. In FIGS. 4A-D, the Y axis represents the K_(D)′ valuesafter 4 months of drug treatment or placebo. In FIGS. 5A and 5B, the Yaxis represents percent change in affinity (K_(D)′ values) over 4 monthsof drug treatment or placebo. FIGS. 6A and 6B reflect similar dataanalysis as shown in FIG. 5 but with an expanded patient population, assummarized in Table 5.

The data for the expanded patient population are summarized in FIG. 7(100 mg/week LJP 394). When results from FIG. 7 are broken down intohigh affinity (Kd′<0.8 mg/ml) and low affinity groups (Kd′>0.8 mg/ml) asshown in FIG. 8, the high affinity group shows change in affinity over a4 month period whereas the low affinity group does not show anysignificant change in affinity.

The change in antibody affinity is also depicted in FIG. 9. The sampleswere selected based on the initial (pre-treatment affinity) andsegregated into three groups of relative affinity: high, medium, and lowaffinities. The affinity values are the mean of 3 patients. The solidtriangles represent “low affinity patients” (Kd′ mean about 1.13 mg/ml);the solid circles represent “medium affinity” patients (Kd′ mean about0.44 mg/ml); and the solid squares represent “high affinity” patients(Kd′ mean about 0.14 mg/ml).

These data demonstrate that patients with high initial affinity for theLJP 394 epitope (low numeric K_(d)′=high affinity) exhibited greaterresponse to the drug over 4 months when compared patients with lowerinitial affinity antibodies. They also suggest that one could preselectpatients most likely to have a positive serological response to LJP 394.

The change over time in anti-ds DNA antibody levels, as assessed bynon-competitive Farr assay in the LJP 394-treated patients was analyzedby stratifying the patients into 2 groups, low affinity (K_(D)′>0.8mg/ml) and high affinity (K_(D)′≦0.8 mg/ml). FIG. 10 depicts acomparison of the two groups. Data collected after high dosecorticosteroid and/or cyclophosphamide treatment was excluded. The lowaffinity group is depicted with a dotted line while the high affinitygroup is depicted with a solid line. The areas with gray, shadedbackground show the dosing periods. The first dosing period, indicatedby the first block of gray background, is the induction period duringwhich patients were given 100 mg/week of LJP 394 or placebo. The white,non-shaded area is the non-dosing period when no LJP 394 or placebo wasadministered. The second dosing period, indicated by the second block ofgray background, was a period during which patients were given 50mg/week of LJP 394 or placebo. As shown in FIG. 10, anti-dsDNA antibodylevels are reduced in the high affinity group by the administration of100 mg/wk and 50 mg/week of LJP 394. The anti-dsDNA antibody levels ofthe low affinity group was reduced somewhat with the administration of100 mg/week of LJP 394 and not reduced with the administration of 50mg/week LJP 394. As shown in FIG. 10, there is a significant reductionof anti-ds DNA antibodies in the high affinity group.

Renal flares. FIG. 11A is similar to the graph in FIG. 5A, except thatpatients that presented with SLE-associated flares are identified bydarkened boxes. FIG. 11B (LJP 394-treated patients) is similar to FIG.11A except that an expanded population of patients has been used foranalysis. FIG. 11C shows the percent change in affinity forplacebo-treated patients. The solid shapes (square for LJP 394-treatedpatients and circle for placebo-treated patients) indicate the patientswho experienced renal flares during the treatment period. The clusteringof lupus flares in the low affinity patients is evident in FIGS. 11 and12. These data indicate that patients with high affinity for LJP 394respond better to the drug by both serological and clinical measures andexperienced fewer renal flares.

As indicated in FIG. 13, there appears to be no effect of placebotreatment on Kd′ over 4 months of administration of placebo. While thenumber of data points is limited in this graph, the lack of effect ofplacebo treatment on Kd′ is identical to the first study.

FIG. 14 depicts a renal flare analysis for high affinity individuals(Kd′ of less than 0.8 mg/ml). The X-axis is time since the first dose ofLJP 394 (or placebo) measured in months and wherein the Y-axis is theproportion of individuals who did not experience a renal flare. A higherproportion of high affinity patients receiving LJP 394 did not have arenal flare as compared to high affinity placebo patients. Forplacebo-treated patients, more flares occurred and these flares occurredearlier in the trial.

Example 4 Dependence on Corticosteroids and/or Cyclophosphamide

High affinity patients (Kd′<0.8 mg/ml) in study 2 (see Example 3), wereanalyzed in terms of time to the first intervention with high dosecorticosteroid and/or cyclophosphamide (HDCC) treatment. The results ofthis analysis are depicted in FIG. 15. The solid line indicates the LJP394-treated individuals while the dotted line indicates theplacebo-treated individuals. The X-axis indicates time since first doseof LJP 394 (or placebo) in terms of months and the Y-axis indicates thepercentage of individuals that did not receive HDCC intervention. HDCCintervention refers to the first intervention with an increased dosageof corticosteroid alone or with cyclophosphamide such that the increasedosage is at least a 15 mg/day increment and such that the total amountof corticosteroid and/or cyclophosphamide treatment is greater than 20mg/day. HDCC may be administered using standard clinical protocols. Aclinician may monitor a patient and determine when HDCC treatment isneeded by evaluating factors including, but not limited to, proteinurialevels, hematuria levels, and serum creatinine levels. In general,patients who experience renal flares are given HDCC treatment. More HDCCintervention was required in high affinity placebo-treated patients thanin high affinity LJP 394-treated patients and the HDCC intervention wasrequired earlier in the patients treated with placebo.

Example 5 Use of LJP 394 Affects Clinical Outcome

Four parameters of clinical outcome: renal flares throughout the trial,renal flares during the induction period, HDCC intervention and majorSLE flares, were analyzed for the high affinity group and the resultsare depicted in FIG. 16. The slashed bars represent high affinityplacebo-treated patients and the solid bars represent LJP 394-treatedhigh affinity (Kd′<0.8 mg/ml) patients. HDCC intervention refers to thefirst incidence of the administration of increased dosagecorticosteroids and/or cyclophosphamide, as defined herein. Major SLEflares include hospitalization due to SLE, first occurrence of high doseprednisone, first occurrence of high dose cyclophosphamide, and/or deathdue to SLE. The induction period during which renal flares were measuredis the first 4 months of the LJP 394 treatment at 100 mg/week (orplacebo). For the duration of the treatment, after the initial inductionperiod, there was intermittent dosing. For the non-induction period, thedosage was 50 mg/week of LJP 394 (or placebo) with periods of notreatment. FIG. 16 illustrates that high affinity patients respondedsignificantly better to LJP 394 treatment than placebo-treated patientsand that the clinical outcome of the high affinity patients is markedlybetter.

In contrast, FIG. 17 shows the entire patient population pool (i.e., the“intent to treat” population) which contains both low and high affinitypatients. The LJP 394-treated patients showed some improvement inclinical outcome over the placebo-treated patients; however, theclinical outcome for this “mixed” LJP 394-treated group is not asstriking compared to the results in high affinity patients (as shown inFIG. 16). Thus, the high affinity patients exhibit more positiveclinical outcomes (i.e., fewer renal flares throughout the trial, fewerrenal flares during the induction period, more time to HDCCintervention, and fewer major SLE flares).

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be, apparent to those skilled in the art thatcertain changes and modifications can be practiced. Therefore, thedescription and examples should not be construed as limiting the scopeof the invention, which is delineated by the appended claims.

1. A method of treating systemic lupus erythematosus (SLE) in anindividual, comprising administering to the individual a conjugatecomprising (a) a non-immunogenic valency platform molecule and (b) twoor more double stranded DNA (dsDNA) epitopes which specifically bind toan antibody from the individual which specifically binds to doublestranded DNA, wherein affinity of the dsDNA epitope for the antibodyfrom the individual is used as a basis for selecting the individual toreceive or continue to receive the treatment.
 2. The method of claim 1,wherein the dsDNA epitopes are polynucleotides.
 3. The method of claim2, wherein the polynucleotides are double stranded DNA.
 4. A method oftreating SLE in an individual, comprising administering to theindividual a conjugate comprising (a) a non-immunogenic valency platformmolecule and (b) two or more polynucleotides which specifically bind toan antibody from the individual which specifically binds to doublestranded DNA, said polynucleotide consisting essentially of the sequence5′-GTGTGTGTGTGTGTGTGTGT-3′, wherein the apparent equilibriumdissociation constant (K_(D)′) for the polynucleotide with respect tothe antibody from the individual before or upon initiation of treatmentis less than about 1.0 mg IgG per ml, and wherein said K_(D)′ value or afunctional equivalent thereof is used as a basis for selecting theindividual to receive the treatment.
 5. The method of claim 4, whereinthe K_(D)′ is less than about 0.8.
 6. The method of claim 4, wherein theK_(D)′ is less than about 0.5.
 7. The method of claim 4, wherein theK_(D)′ is less than about 0.2.
 8. The method of claim 4, wherein theplatform molecule is

wherein PN is the polynucleotide.
 9. The method of claim 5, wherein thepolynucleotide consists of the sequence 5′-GTGTGTGTGTGTGTGTGTGT-3′. 10.The method of claim 9, wherein the platform molecule is

wherein PN is the polynucleotide.
 11. The method of claim 9, wherein theK_(D)′ is less than about 0.5.
 12. A method of treating SLE in anindividual comprising: (a) assessing affinity of an anti-double strandedDNA antibody from the individual with respect to a dsDNA epitope whichis to be used in treatment, wherein the individual is selected fortreatment based on said antibody affinity; and (b) administering to saidselected individual a conjugate comprising (a) a non-immunogenic valencyplatform molecule and (b) two or more of the dsDNA epitopes.
 13. Themethod of claim 12, wherein the dsDNA epitope is a polynucleotide. 14.The method of claim 13, wherein the polynucleotide is double strandedDNA. 15-64. (canceled)